Protease variants

ABSTRACT

The present invention relates to methods for producing variants of a parent RP-II protease and the variants having altered properties as compared to the parent RP-II protease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 10/588,555 filed on Jun. 1, 2007 which is a 35 U.S.C. 371 national application of PCT/DK2005/000097 filed Feb. 14, 2005 which claims priority or the benefit under 35 U.S.C. 119 of Danish Application no. PA 2004 00226 filed Feb. 13, 2004 and U.S. provisional application No. 60/558,191 filed Mar. 31, 2004, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to variants of proteases belonging to the RP-II or C-component type, and methods for the construction of such variants with altered properties, such as stability (e.g. thermostability or storage stability), Ca²⁺ dependency, and pH dependent activity.

BACKGROUND OF THE INVENTION

Enzymes have been used within the detergent industry as part of washing formulations for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellulases, hemicellulases or mixtures of enzymes are also often used. Proteases are also used in other fields, such as production of diary products, processing of hides, feed processing, etc.

To improve the cost and/or the performance of proteases there is an ongoing search for proteases with altered properties, such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca²⁺ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.), modified specificity in respect of substrates, etc.

The search for proteases with altered properties includes both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g. genetic manipulation of the nucleic acid sequence encoding said proteases. Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific property of the protein.

One group of proteases, which has been indicated for use in detergents, food processing, feed processing is the RP-II proteases or C-component proteases belonging to the protease family S1B, glutamic-acid-specific endopeptidases. This family has till now only received relatively minor attention and has not been further grouped into different sub-groups. However, from the amino acid identities of isolated RP-II proteases it is evident that subgroups exist. Bacillus proteases of the RP-II type are serine proteases that in primary structure are similar to chymotrypsin.

The first description of a protease of the RP-II family of Bacillus proteases was in U.S. Pat. No. 4,266,031 (Tang et al., Novo Industri A/S), where it was designated Component C and tentatively (and incorrectly) characterised as not being a serine protease or metallo protease. Component C was considered a contaminant in the production of the Bacillus licheniformis alkaline protease, subtilisin Carlsberg.

In EP 369 817 (Omnigene Bioproducts, Inc.) the B. subtilis member of the RP-II family was identified by its amino acid and DNA sequences. The enzyme was again stated not to be a serine protease, and the family name RP-II designated (Residual Protease II). The enzyme was characterized further as a metallo protease by the inventors of EP 369 817 (Rufo et al., 1990, J. Bacteriol. 2 1019-1023, and Sloma et al., 1990, J. Bacteriol. 172 1024-1029), designating the enzyme as mpr.

In WO 91/13553 (Novozymes A/S) the amino acid sequence of the C component was disclosed, stating that it is a serine protease specific for glutamic and aspartic acid, while EP 482 879 (Shionogi & Co. Ltd.) disclosed the enzyme and a DNA sequence encoding the C component from B. licheniformis ATCC No. 14580, naming the enzyme BLase. In EP 482 879 the protease is described as being specific for glutamic acid (see also Kakudo et al. “Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580”. J. Biol. Chem. 267:23782 (1992)).

In 1997 Okamoto et al. (Appl. Microbiol. Biotechnol. (1997) 48 27-33) found that the B. subtilis homologue of BLase, named BSase was identical to the above-mentioned enzyme, mpr/RP-II.

In 1999 Rebrikov et al. (Journal of Protein Chemistry, Vol. 18, No. 1, 1999) disclosed a Glu-specific protease from B. intermedius that also belongs to the RP-II family.

In WO 01/16285 a number of further RP-II protease were disclosed with DNA and amino acid sequences. These RP-II proteases were isolated from B. pumilus, B. halmapalus and B. licheniformis. WO 01/16285 also discloses a number of variants of RP-II proteases. These variants were based on various concepts relating to the primary structure of the RP-II proteases (amino acid sequences).

The homology matrix in Table 1 below clearly indicates that the RP-II proteases 1 to 8 are a distinct group of Glu-specific proteases that are clearly different from the other Glu-specific proteases in the Matrix

TABLE 1 1 2 3 4 5 6 7 8 9 10 11 12 13 1 100 99 97 60 55 55 47 59 46 45 45 47 49 2 100 99 60 60 59 50 61 50 44 45 46 52 3 100 60 57 54 47 60 47 45 45 44 49 4 100 94 92 68 57 44 38 40 42 47 5 100 91 59 54 44 42 40 43 45 6 100 63 53 39 42 46 41 45 7 100 48 41 41 40 36 44 8 100 50 45 46 46 54 9

 

 

 

 

10

 

 

 

11

 

 

12

 

13

In the matrix the sequences are identified by the patent publication in which first published or sequence database accession numbers. 1. Bacillus sp. JA96 glutamic-acid-specific endopeptidase, JA96, WO 01/16285 2. 1p3e B. Intermedius, glutamic-acid-specific endopeptidase, BIP, EMBL No. Y5136, Rebrikov et al., Journal of Protein Chemistry, Vol. 18, No. 1, 1999 3. Bacillus sp. BO32 glutamic-acid-specific endopeptidase, BO32, WO 01/16285 4. Bacillus licheniformis, BLC, WO 01/16285 (cf. U.S. Pat. No. 4,266,031) 5. Bacillus sp. CDJ31 glutamic-acid-specific endopeptidase, CDJ31, WO 01/16285 6. Bacillus sp. AC116 glutamic-acid-specific endopeptidase, AC116, WO 01/16285 7. mpr_bacsu Bacillus subtilis serine protease, MPR, EP 369 817 8. Bacillus sp. AA513 glutamic-acid-specific endopeptidase, AA513, WO 01/16285 9. eta_staau Staphylococcus aureus exfoliative toxin A (Lee et al. Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus; J. Bacteriol. 169: 3904 (1987)) 10. etb_staau Staphylococcus aureus exfoliative toxin B (Jackson, M. P.; Iandolo, J. J.; Sequence of the exfoliative toxin B gene of Staphylococcus aureus; J. Bacteriol. 167: 726 (1986)) 11. q53781 Staphylococcus aureus (strain Mu50/ATCC 700699) (Rieneck et al.; Submitted (JUN-1996) to the EMBL/GenBank/DDBJ databases) 12. q53782 Staphylococcus aureus (strain Mu50/ATCC 700699) (Rieneck et al., “Molecular cloning and expression of a novel Staphylococcus aureus antigen”. Biochim. Biophys. Acta 1350: 128 (1997) 13. stsp_staau Staphylococcal serine endoproteinase V8 Glu-C (Gray, “Nucleotide sequence of the serine protease gene of Staphylococcus aureus, strain V8” Nucleic Acids Res. 15: 6757 (1987)

The three-dimensional structure of the protease Toxin A from Staphylococcus aureus. Belonging to the S1B family has been determined by Cavarelli, J., et al. Structure Vol. 5, p. 813 1997.

However, despite the sequence homology between the proteases belonging to the RP-II proteases and Toxin A from Staphylococcus aureus, modelling of the three-dimensional structure of RP-II proteases on the basis of the three-dimensional structure of Toxin A from Staphylococcus aureus may result in an incorrect three-dimensional structure because of structural differences, especially because the distinct difference in sequence homology to the RP-II proteases.

The inventors of the present invention have elucidated the three-dimensional structure of the C-component protease from Bacillus licheniformis and found that there are several differences between this and the three-dimensional structure of Toxin A from Staphylococcus aureus also belonging to the S1B subgroup of proteases. This surprising difference in structure makes it advantageous to use the BLC structure as basis for homology modelling of RP-II proteases, which, in turn, will improve the ability to obtain desired changes in functionality by protein engineering.

BRIEF DESCRIPTION OF THE INVENTION

The inventors have modified the amino acid sequence of a RP-II protease to obtain variants with improved properties, based on the three-dimensional structure of the C-component. The variants will have altered properties, such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca²⁺dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.

Accordingly, the object of the present invention is to provide a method for constructing RP-II proteases having altered properties, in particular to provide a method for constructing RP-II proteases having altered properties as described above.

Thus, in its broadest aspect, the present invention relates to a method for constructing a variant of a parent RP-II protease, wherein the variant has at least one altered property as compared to said parent RP-II protease, which method comprises:

i) analyzing the three-dimensional structure of the RP-II protease to identify, on the basis of an evaluation of structural considerations, at least one amino acid residue or at least one structural region of the RP-II protease, which is of relevance for altering said property; ii) constructing a variant of the RP-II protease, which as compared to the parent RP-II protease, has been modified in the amino acid residue or structural part identified in i) so as to alter said property; and iii) testing the resulting RP-II protease variant for said property.

Although it has been described in the following that modification of the parent RP-II protease in certain regions and/or positions is expected to confer a particular effect to the thus produced RP-II protease variant, it should be noted that modification of the parent RP-II protease in any of such regions may also give rise to any other of the above-mentioned effects. For example, any of the regions and/or positions mentioned as being of particular interest with respect to, e.g., improved thermostability, may also give rise to, e.g., higher activity at a lower pH, an altered pH optimum, or increased specific activity, such as increased peptidase activity.

Further aspects of the present invention relates to variants of a RP-II protease, the DNA encoding such variants and methods of preparing the variants. Still further aspects of the present invention relates to the use of the variants for various industrial purposes, in particular as an additive in detergent compositions. Other aspects of the present invention will be apparent from the below description as well as from the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a schematic structure of the RP-II protease from Bacillus licheniformis, BLC (SEQ ID NO: 2).

FIG. 2 shows a 3D structure based alignment of the wild type RP-II proteases 1 to 8 of Table 1 (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16).

FIG. 3 shows the BLC protease ribbon structure in black, with indication of active site residues, the bound peptide and the ion-binding site. The calcium ion is the sphere at the bottom of the Figure, the active site residues are in light grey and shown in stick model, and the bound peptide DAFE is in medium grey and shown in stick model.

BRIEF DESCRIPTION OF APPENDICES

APPENDIX 1 provides the structural coordinates for the solved crystal 3D structure of the BLC RP-II protease, in the standard pdb format. The residues are numbered from 1-217, the calcium ion is numbered 301, and the DAFE substrate is numbered 401-404.

DEFINITIONS

Prior to discussing this invention in further detail, the following terms and conventions will first be defined.

For a detailed description of the nomenclature of amino acids and nucleic acids and modifications introduced in a polypeptide or protein and especially in a RP-II protease by genetic manipulation, we refer to WO 01/16285 pages 5 to 15, hereby incorporated by reference.

The term “RP-II proteases” refers to a sub-group of serine protease, belonging to the protease family S1B, glutamic-acid-specific endopeptidases. Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the RP-II proteases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue.

The RP-II proteases have a homology to the rest of the S1B protease family of around 50% (using the UWGCG version 8 software GAP program), or more preferred a homology higher than 55%. Table 1 demonstrate homologies between various S1B proteases. The RP-II proteases, nos. 1 to 8, are in Table 1 indicated in bold and the other S1B proteases, nos. 9 to 13, in bold italics. Table 1 shows that there is a clear distinction to the RP-II proteases from the other S1B proteases, but it is also clear that among the RP-II proteases there are subgroups. One subgroup comprises nos. 1, 2, and 3; and another subgroup comprises nos. 4, 5, and 6. The lengths of the listed RP-II proteases vary from 215 to 222 amino acid residues and experience within the subtilisin subgroups of subtilases indicates that such a variation in length probably has only little effect on the 3-dimensional structures of these and other RP-II protease subgroups.

Parent

The term “parent” is in the context of the present invention to be understood as a protein, which is modified to create a protein variant. The parent protein may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means. For instance, the parent protein may be a variant of a naturally occurring protein which has been modified by substitution, chemical modification, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide. Thus the term “parent RP-II protease” refers to a RP-II protease which is modified to create a RP-II protease variant.

Variant

The term “variant” is in the context of the present invention to be understood as a protein which has been modified as compared to a parent protein at one or more amino acid residues.

Modification

The term “modification(s)” or “modified” is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein. The modification(s) may be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest. Thus the term “modified protein”, e.g. “modified RP-II protease”, is to be understood as a protein which contains modification(s) compared to a parent protein, e.g. RP-II protease.

Homology

“Homology” or “homologous to” is in the context of the present invention to be understood in its conventional meaning and the “homology” between two amino acid sequences should be determined by use of the “Similarity” parameter defined by the GAP program from the University of Wisconsin Genetics Computer Group (UWGCG) package using default settings for alignment parameters, comparison matrix, gap and gap extension penalties. Default values for GAP penalties, i.e. GAP creation penalty of 3.0 and GAP extension penalty of 0.1 (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711). The method is also described in S. B. Needleman and C. D. Wunsch, Journal of Molecular Biology, 48, 443-445 (1970). Identities can be extracted from the same calculation. The homology between two amino acid sequences can also be determined by “identity” or “similarity” using the GAP routine of the UWGCG package version 9.1 with default setting for alignment parameters, comparison matrix, gap and gap extension penalties can also be applied using the following parameters: gap creation penalty=8 and gap extension penalty=8 and all other parameters kept at their default values. The output from the routine is besides the amino acid alignment the calculation of the “Percent Identity” and the “Similarity” between the two sequences. The numbers calculated using UWGCG package version 9.1 is slightly different from the version 8.

Naming of RP-II Proteases

In describing the RP-II proteases of the invention the following abbreviations are used for ease of reference:

BLC=RP-II protease from Bacillus licheniformis (U.S. Pat. No. 4,266,031), AA513=RP-II protease from Bacillus halmapalus AA513 (WO 01/16285), AC116=RP-II protease from Bacillus licheniformis AC116 (WO 01/16285) BO32=RP-II protease from Bacillus pumilus BO32 (WO 01/16285), CDJ31=RP-II protease from Bacillus licheniformis CDJ31 (WO 01/16285), JA96=RP-II protease from Bacillus pumilus JA96 (WO 01/16285), MPR=RP-II protease from Bacillus subtilis IS75 (EP 369 817 B1) BIP=RP-II protease from B. intermedius (Rebrikov et al., Journal of Protein Chemistry, Vol. 18, No. 1, 1999)

Sequence Listing

In the appended Sequence Listing the RP-II proteases are indicated as:

SEQ. ID. NO. 1=BLC (DNA), SEQ. ID. NO. 2=BLC (AA), SEQ. ID. NO. 3=AA513 (DNA), SEQ. ID. NO. 4=AA513 (AA), SEQ. ID. NO. 5=AC116 (DNA), SEQ. ID. NO. 6=AC116 (AA) SEQ. ID. NO. 7=BO32 (DNA), SEQ. ID. NO. 8=BO32 (AA) SEQ. ID. NO. 9=CDJ31 (DNA), SEQ. ID. NO. 10=CDJ31 (AA) SEQ. ID. NO. 11=JA96 (DNA), SEQ. ID. NO. 12=JA96 (AA) SEQ. ID. NO. 13=BSMPR (DNA), SEQ. ID. NO. 14=BSMPR (AA) SEQ. ID. NO. 15=BIP (DNA), SEQ. ID. NO. 16=BIP (AA) Position

The term “position” is in the context of the present invention to be understood as the number of an amino acid residue in a peptide, polypeptide or protein when counting from the N-terminal end of said peptide/polypeptide. The position numbers used here normally refer directly to different RP-II proteases.

The RP-II proteases are numbered individually according to each of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, and 16.

Corresponding Position

The invention, however, is not limited to variants of these particular RP-II proteases but extends to parent proteases containing amino acid residues at positions which are “equivalent” to the particular identified residues in Bacillus licheniformis RP-II protease. In some preferred embodiment of the present invention, the parent protease is JA96 or BIP RP-II protease and the substitutions are made at the equivalent amino acid residue positions in JA96 or BIP corresponding to those listed above.

A residue (amino acid) position of a RP-II protease is equivalent to a residue (position) of the Bacillus licheniformis RP-II protease if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in Bacillus licheniformis RP-II protease (i.e., having the same or similar functional capacity to combine, react, or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor protease is directly compared to the Bacillus licheniformis RP-II protease, BLC, primary sequence by aligning the amino acid sequence of an isolated or parent wild type enzyme with a suitable well-known enzyme of the same group or class of enzymes defines a frame of reference. This type of numbering was used in WO 01/16285.

If nothing else is indicated herein, in the present instance the Bacillus licheniformis RP-II protease, first designated component C and therefore here abbreviated BLC, has been chosen as standard.

In order to establish homology to the tertiary structure (3D structure) of BLC, the 3D structure based alignment in FIG. 2 has been provided. By using this alignment the amino acid sequence of a precursor RP-II protease may be directly correlated to the Bacillus licheniformis RP-II protease, BLC, primary sequence. For a novel RP-II protease sequence, the (3D based) position corresponding to a position in BLC is found by

-   -   i) identifying the RP-II protease from the alignment of FIG. 2         that is most homologous to the novel sequence,     -   ii) aligning the novel sequence with the sequence identified to         find the corresponding position in the RP-II protease from FIG.         2, and     -   iii) establishing from FIG. 2 the corresponding position in BLC.

For comparison and finding the most homologous sequence the GAP program from GCG package as described below are used.

The alignment can as indicated above be obtained by the GAP routine of the GCG package version 8 to number the variants using the following parameters: gap creation penalty=3 and gap extension penalty=0.1 and all other parameters kept at their default values.

The alignment of FIG. 2 defines a number of deletions and insertions in relation to the sequence of BLC. In the alignment deletions are indicated by asterixes (*) in the referenced sequence, and the referenced enzyme will be considered to have a gap at the position in question. Insertions are indicated by asterixes (*) in the BLC sequence, and the positions in the referenced enzyme are given as the position number of the last amino acid residue where a corresponding amino acid residue exists in the standard enzyme with a lower case letter appended in alphabetical order, e.g. 82a, 82b, 82c, 82d, see FIG. 2.

In case the referenced enzyme contains a N- or C-terminal extension in comparison to BLC; an N-terminal extension is given the position number 0a, 0b, etc. in the direction of the N-terminal; and a C-terminal extension will be given either the position number of the C-terminal amino acid residue of BLC with a lower case letter appended in alphabetical order, or simply a continued consecutive numbering.

Thus for comparisons RP-II proteases are numbered by reference to the positions of the BLC RP-II protease (SEQ ID NO: 2) as provided in FIG. 2. The position is then indicated as “corresponding to BLC”.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have elucidated the three-dimensional structure of BLC, SEQ ID NO:2 by X-ray crystallography and found that there are several interesting features in the structure of this protease in comparison with the known structures of other proteases, such as the RP-II proteases. These features include both similarities and differences.

RP-II Proteases

As described above a RP-II protease is in the context of the present invention to be understood as a protease which has at least 50% homology to BLC (SEQ ID NO:2). In particular said protease may have at least 55% homology to BLC, i.e. to SEQ ID NO:2. The invention thus relates to variant RP-II proteases having at least 50% homology to BLC.

Specifically the variants of the invention may comprise RP-II proteases comprising a number of modifications or modifications in a number of positions ranging from at least one and up to 50, or from 1 to 45, or from 1 to 40, or from 1 to 35, or from 1 to 30, or from 1 to 25, or from 1 to 20, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2 modifications or positions. Such modifications comprising substitutions, deletions and insertions in the indicated number or number of positions.

A RP-II protease variant of the present invention is encoded by an isolated polynucleotide, which nucleic acid sequence has at least 50% homology with the nucleic acid sequence shown in SEQ ID NO: 1, and where the polynucleotide encodes a variant RP-II protease in relation to a parent protease.

In a first embodiment of the present invention a RP-II protease suitable for the purpose described herein may be a RP-II protease homologous to the three-dimensional structure of BLC, i.e. it may be homologous to the three-dimensional structure defined by the structure coordinates in Appendix 1 by comprising the structural elements defined below.

It is well-known to a person skilled in the art that a set of structure coordinates for a protein or a portion thereof is a relative set of points that define a shape in three dimensions; it is possible that an entirely different set of coordinates defines an identical or a similar shape. Moreover, slight variations in the individual coordinates may have little or no effect on the overall shape.

These variations in coordinates may be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates of Appendix 1 (BLC structure) may be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above. Alternatively, said variations may be due to differences in the primary amino acid sequence.

When such variations are within an acceptable standard error as compared to the structure coordinates of Appendix 1 said three-dimensional structure is within the context of the present invention to be understood as being homologous to the structure of Appendix 1. The standard error may typically be measured as the root mean square deviation of e.g. conserved backbone residues, where the term “root mean square deviation” (RMS) means the square root of the arithmetic mean of the squares of the deviations from the mean.

It is also well-known to a person skilled in the art that within a group of proteins which have a homologous structure there may be variations in the three-dimensional structure in certain areas or domains of the structure, e.g. loops, which are not, or at least only of a small importance to the functional domains of the structure, but which may result in a big root mean square deviation of the conserved residue backbone atoms between said structures.

Thus it is well known that a set of structure coordinates is unique to the crystallised protein. No other three dimensional structure will have the exact same set of coordinates, be it a homologous structure or even the same protein crystallised in different manner. There are natural fluctuations in the coordinates. The overall structure and the inter-atomic relationship can be found to be similar. The similarity can be discussed in terms of root mean square deviation of each atom of a structure from each “homologous” atom of another structure. However, only identical proteins have the exact same number of atoms. Therefore, proteins having a similarity below 100% will often have a different number of atoms, and thus the root mean square deviation can not be calculated on all atoms, but only the ones that are considered “homologous”. A precise description of the similarity based on the coordinates is thus difficult to describe and difficult to compute for homologous proteins. Regarding the present invention, similarities in 3D structure of different RP-II proteases can be described by the content of homologous structural elements, and/or the similarity in amino acid or DNA sequence

Examples of BLC like RP-II proteases include the BLC=RP-II protease from Bacillus licheniformis (cf. U.S. Pat. No. 4,266,031), AA513=RP-II protease from Bacillus halmapalus AA513 (NP000368), AC116=RP-II protease from Bacillus licheniformis AC116 (NP000364), BO32=RP-II protease from Bacillus pumilus BO32 (NP000366), CDJ31=RP-II protease from Bacillus licheniformis CDJ31 (NP000365), JA96=RP-II protease from Bacillus pumilus JA96 (NP000367), MPR=RP-II protease from Bacillus subtilis IS75 (cf. EP 369 817 B1), BIP=RP-II protease from B. intermedius (EMBL No. Y5136, Rebrikov et al., Journal of Protein Chemistry, Vol. 18, No. 1, 1999)

Accordingly, a preferred embodiment of the present invention is a variant of a parent RP-II protease or a RP-II protease variant which is at least 50% homologous to the sequence of SEQ ID NO 2 preferably at least 55%, preferably at least 65%, at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14 or 16.

A further embodiment of the invention is a RP-II protease variant comprising the following structural characteristics:

-   -   a) two beta-barrel domains each comprising six long strands in         antiparallel organisation,     -   b) three alpha helices,     -   c) at least one ion-binding site,     -   d) an active site comprising the amino acid residues His, Asp         and Ser.

The potential ion binding site is defined as similar coordination or arrangement of the coordinates as in the 3D structure of BLC having one calcium ion coordinated by the Ile 3 carbonyl atom O, the Ser 5 carbonyl atom O and bidendate by the Asp 161 Carboxyl acid group and the further coordination made by waters. The calcium may be substituted in the structure by water but then having the same coordination.

The RP-II protease variants of the present invention are encoded by isolated polynucleotides, which nucleic acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15, and where the polynucleotide encodes a variant RP-II protease in relation to a parent protease.

Further the isolated nucleic acid sequence encoding a RP-II protease variant of the invention hybridizes with a complementary strand of the nucleic acid sequence shown in SEQ ID NO: 1 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.

Suitable experimental conditions for determining hybridization at low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5×SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5×SSC, 5×Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of 10 ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), ³²P-dCTP-labeled (specific activity>1×10⁹ cpm/μg) probe for 12 hours at ca. 45° C. The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS at least 55° C. (low stringency), more preferably at least 60° C. (medium stringency), still more preferably at least 65° C. (medium/high stringency), even more preferably at least 70° C. (high stringency), and even more preferably at least 75° C. (very high stringency).

Three-Dimensional Structure of RP-II Proteases

The BLC RP-II protease was used to elucidate the three-dimensional structure forming the basis for the present invention.

The structure of BLC was solved in accordance with the principle for x-ray crystallographic methods, for example, as given in X-Ray Structure Determination, Stout, G. K. and Jensen, L. H., John Wiley & Sons, Inc. NY, 1989.

The structural coordinates for the solved crystal structure of BLC are given in standard PDB format (Protein Data Bank, Brookhaven National Laboratory, Brookhaven, Conn.) as set forth in Appendix 1. It is to be understood that Appendix 1 forms part of the present application. In the context of Appendix 1, the following abbreviations are used: CA refers to c-alpha (carbon atoms) or to calcium ions, (however to avoid misunderstandings we normally use the full names “c-alpha atoms”, “calcium” “Ca” or “ion” in the present specification). Amino acid residues are given in their standard three-letter code or the standard one-letter code. The structural coordinates in Appendix 1 contain the protease structure wherein the active serine was replaced by alanine and a complex formed with the peptide DAFE (=Asp-Ala-Phe-Glu) as well as water molecules. The protease coordinates has a chain identification called A, whereas the peptide is called B, the calcium ion is called C, and the water is W. In the following the positions of the mentioned residues refer to the sequence of BLC as disclosed in SEQ ID NO: 2.

The overall structure of BLC falls into the 51 group of the proteases (MEROPS). The structure is a trypsin type of fold with two beta-barrel domains. The beta-barrel's each consists of six antiparallel beta-sheets folded into a beta-barrel. The topology can be described as 51-S2-S3-S6-S5-S4 for the strands in both beta-barrels. It is assumed that all the RP-II proteases fall within the same general overall structure.

The 3D structure of C-component serine protease from Bacillus licheniformis has 16 strands of which the 12 bigger strands compose the two beta-barrels; and 3 helixes. The four very short strands are number 1, 5, 6 and 10 counting from the N-terminal and are composed of residue numbers 9-10, 50-51, 56-57 and 114-115. The other strands are residue numbers 22-26, 31-36, 41-44, 62-65, 77-83, 99-102, 126-131, 142-151, 156-159, 171-177, 182-192 and 201-205. One main helix C-terminal residue number 208-219. Two very small helices are composed of residues 86-90 and 106-110.

The active site consists of a triad involving the Ser in position 167, the His in position 47, and the Asp in position 96.

The 3D structure of BLC has one calcium ion coordinated by the carbonyl oxygen atom of Ile in position 3, the carbonyl oxygen atom of Ser in position 5, and bidendate by the Carboxylic acid group of Asp in position 161. Further coordinations are made by water molecules.

The calcium ion is placed in a distance from the CA atoms of the active site and Gly in position 168 as provided below:

Ser 167 CA atom to Ca ion: 16.07 Å His 47 CA atom to Ca ion: 24.27 Å Asp 96 CA atom to Ca ion: 23.72 Å Gly 168 CA atom to Ca ion: 19.20 Å

The position of an ion-binding site can be defined by the distance to four specific atoms in the core structure. The distance from the ion-binding site to the c-alpha atoms of the three active site residues has been chosen. Throughout the RP-II proteases the residues Ser, His and Asp in the active site are highly conserved. In BLC they are Asp96, His47 and Ser167. The fourth distance chosen is the distance to the c-alpha atom of the amino acid residue coming first after the active site serine residue in the sequence (herein after called “next to Ser”); in the 3D structure of BLC it is Gly168.

In a preferred embodiment of the present invention, the distance between the ion-binding site and i) Asp c-alpha atom is 22.50-24.00 Å, ii) His c-alpha atom is 23.25-25.25 Å, iii) Ser c-alpha atom is 15.00-17.00 Å, iv) next to Ser c-alpha atom is 18.20-20.20 Å,

However these distances may vary from one RP-II protease to the other, and as described above, the ion binding site may also bind to a sodium ion. The present distances are given with a calcium ion in the structure. If a sodium ion was bound instead the distances would be shifted a little bit. Generally the distances can vary ±0.8 Å, preferably ±0.7 Å, ±0.6 Å, ±0.5 Å, ±0.4 Å, or most preferably ±0.3 Å.

Further, in the RP-II proteases, the peptide structure circumscribing the ion-binding site is composed of the amino acid residues placed in positions 1-7, 159-162 and 143-145 with the coordinating atoms being the backbone carbonyl oxygen atom of residues 13, S5, D161 and water molecules.

3D structures of RP-II proteases can be modelled using the known structure of a related protease and general modelling tools as shown in Example 1. A prerequisite for obtaining a realistic 3D model structure is that the model is based on an adequate sequence homology higher than 50%, preferably higher than 55%, and even more preferred higher than 60% to the sequence of the protease for which the structure is known. RP-II Protease models can be constructed based on the 3D guided sequence alignments to BLC in FIG. 2.

Therefore 3D structure models of RP-II proteases could in principle be made by using the modelling tools and the known 3D structure of the toxin A protease from Staphylococcus aureus from the Exf family of proteases (Cavarelli et al. (1997) The Structure of Staphylococcus aureus Epidermolytic Toxin A, an atypic serine protease, at 1.7 Å resolution, Structure, Vol. 5, p. 813 (pdb name 1ARP).

If compared to the structure of the toxin A protease from Staphylococcus aureus, the structure of the RP-II proteases, as represented by BLC, can be divided into a “common protease” region, an “intermediate” region and a “nonhomologous” region.

The active site can be found in the common protease region, which is structurally closely related to the Toxin A structure. The common protease region is composed of residues 58, 70-83. The common protease region has an RMS lower than 1.2.

Outside the common protease region the structure of the RP-II protease BLC differs from the Toxin A structure to a greater extent.

The intermediate region consists of residues 14-28, 29-51, 94-104, 155-175. The intermediate region has an RMS bigger than 1.2 and less than 1.8. Any relationships between the three-dimensional structure and functionality based on modelling from the S. aureus 3D structure are potentially difficult to predict in this region of the RP-II proteases.

The common region and the intermediate region consist of the majority of the two central beta-barrels, especially the strands of the beta-barrels.

The nonhomologous region consists of residues 1-6, 7-13, 52-57, 59-69, 84-88, 89-93, 105-153. The nonhomologous region has a RMS higher than 1.5. Any relationships between the three-dimensional structure and functionality based on modelling from the S. aureus 3D structure are very difficult to predict in this region of the RP-II proteases.

Inferred structure-function relationships based on model building of a RP-II protease 3D structure on the 3D structure of S. aureus Toxin A would thus be very uncertain and speculative.

Homology Building of RP-II Proteases

A model structure of a RP-II protease can be built using the BLC structure in Appendix 1, or a structure similar to the BLC structure comprising the structural elements (a) two beta-barrel domains each comprising six long strands in antiparallel organisation, (b) three alpha helices, (c) at least one low affinity ion-binding site, and (d) an active site comprising the amino acid residues His, Asp and Ser, or other 3D RP-II protease structures, e.g. established by X-ray structure determination, that may become available in the future, and the Homology™ program or a comparable program, e.g., Modeller™ (both from Molecular Simulations, Inc., San Diego, Calif.). The principle is to align the amino acid sequence of a protein for which the 3D structure is known with the amino acid sequence of a protein for which a model 3D structure has to be constructed. The structurally conserved regions can then be built on the basis of consensus sequences. In areas lacking homology, loop structures can be inserted, or sequences can be deleted with subsequent bonding of the necessary residues using, e.g., the program Homology. Subsequent relaxation and optimization of the structure should be done using either Homology or another molecular simulation program, e.g., CHARMm™ from Molecular Simulations.

Methods for Designing BLC and RP-II or Sib Family Protease Variants

Comparisons of the molecular dynamics of different proteins can give a hint as to which domains are important or connected to certain properties pertained by each protein.

The present invention comprises a method of producing a variant of a parent BLC like RP-II protease, the variant having at least one altered property as compared to the parent BLC like RP-II protease, the method comprising:

-   -   a) producing a model structure of the parent BLC like RP-II         protease on the three-dimensional structure of BLC,     -   b) comparing the model three-dimensional structure of the parent         BLC like RP-II protease to the BLC structure by superimposing         the structures through matching the active residues CA, CB, C,         O, and N atoms,     -   c) identifying on the basis of the comparison in step a) at         least one structural part of the parent BLC like RP-II protease,         wherein an alteration in said structural part is predicted to         result in an altered property;     -   d) modifying the nucleic acid sequence encoding the parent BLC         like RP-II protease to produce a nucleic acid sequence encoding         deletion or substitution of one or more amino acids at a         position corresponding to said structural part, or an insertion         of one or more amino acid residues in positions corresponding to         said structural part;     -   e) expressing the modified nucleic acid sequence in a host cell         to produce the variant RP-II protease;     -   f) isolating the produced protease;     -   g) purifying the isolated protease and     -   h) recovering the purified RP-II protease.

Stability—Alteration of Ion-Binding Site

An ion-binding site is a significant feature of an enzyme. Therefore alterations of the amino acid residues close to the ion-binding site are likely to result in alterations of the stability of the enzyme. Especially modifications affecting the charge distribution and/or the electrostatic field strength at or in the vicinity of the site are important.

Improved Stability

Stabilisation of the ion-binding site of RP-II proteases may be obtained by modifications in positions close to the ion binding site.

Such modifications may comprise the substitution of a positively charged amino acid residue with a neutral or negatively charged residue, or the substitution of a neutral residue with a negatively charged residue or the deletion of a positively charged or neutral residue in positions close to the ion binding site.

Positions located at a distance of 10 Å or less to the ion-binding site of BLC are: 1, 2, 3, 4, 5, 6, 7, 8, 143, 144, 145, 146, 158, 159, 160, 161, 162, 194, 199, 200, and 201. Especially positions 2, 3, 4, 5, 6, 7, 144, 159, 160, 161 located at a distance of 6 Å or less from the ion binding site are important.

Corresponding positions in other RP-II proteases may be identified using FIG. 2 herein.

The modifications D7E and D7Q in BLC are examples of suitable modifications in one of these positions.

Removal of Ion-Binding Site in BLC

By removing the ion-binding site it is possible to alter the dependency of the enzyme on calcium or other ions in the solution.

Removal of the Calcium site in BLC can be done by the substitutions H144R and/or D161R,K+H144Q,N (SEQ ID NO: 2). Similar modifications may be made in structurally corresponding residues in other RP-II proteases.

Alteration of Thermostability

A variant with improved stability (typically increased thermostability) may be obtained by modification of the mobility of identified regions, such as by introduction of disulfide bond(s), substitution with proline, alteration of hydrogen bond contact(s), altering charge distribution, introduction of salt bridge(s), filling in internal structural cavities with one or more amino acids with bulkier side groups (in e.g. regions which are structurally mobile), substitution of histidine residues with other amino acids, removal of a deamidation sites, or by helix capping.

Regions with Increased Mobility:

The below indicated regions of BLC have an increased mobility in the crystal structure of the enzyme, and it is presently believed that these regions can be responsible for stability or activity of BLC and the other RP-II proteases. Especially thermostabilisation may be obtained by altering the highly mobile regions. Generally, thermostability may be improved by making these regions less mobile. Improvements of the enzyme may be obtained by making modifications in the regions and positions identified below. Introducing e.g. larger residues or residues having more atoms in the side chain could increase the stability, or, e.g., introduction of residues having fewer atoms in the side chain could be important for the mobility and thus the activity profile of the enzyme. The regions can be found by analysing the B-factors taken from the coordinate file in Appendix 1, and/or from molecular dynamics calculations of the isotropic fluctuations. These can be obtained by using the program CHARMm from MSI (Molecular Simulations Inc.).

Molecular dynamics simulation at 300K and 400K of BLC reveals the following highly mobile regions:

26-31, 50-55, 89-91, and 193-198, and 4-5, 11-12, 26-31, 50-55, 69-70, 89-91, 178-183, 195-199 and 216-221, respectively.

It is contemplated that modifications in these regions may influence the thermostability of RP-II proteases. Modifications are preferably made in the regions 26-31 (26, 27, 28, 29, 30, 31); 89-91 (89, 90, 91); 216-221 (216, 217, 218, 219, 220, 221), and especially in BLC the substitutions G30A and G91A. Similar modifications may be made in structurally corresponding residues in other RP-II proteases.

Also B-factors (see “in X-Ray Structure Determination, Stout, G. K. and Jensen, L. H., John Wiley & Sons, Inc. NY, 1989”) from crystallographic data indicate the following more mobile regions in the BLC (RP-II protease) structure:

51-56, (i.e. 51, 52, 53, 54, 55, 56) 88-94, (i.e. 88, 89, 90, 91, 92, 93, 94)

118-122 (I. e. 118, 119, 120, 121, 122)

173-183 (i.e. 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183)

It is contemplated that modifications in these regions may influence the thermostability of RP-II proteases. Modifications are preferably made in the regions 51-56 and 118-122.

Disulfide Bonds:

A RP-II protease variant of the present invention with improved stability, e.g. thermostability, as compared to the parent RP-II protease may be obtained by introducing new interdomain or intra-domain bonds to provide a more rigid and stable structure, such as by establishing inter- or intra-domain disulfide bridges. This is done by introducing cysteines in appropriate positions in the RP-II molecule by substitution(s) or insertion(s).

According to the guidelines mentioned above the below mentioned amino acid residues identified in the amino acid sequence of SEQ ID NO: 2 are contemplated as being suitable for cysteine replacement. With one or more of these substitutions with cysteine, disulfide bridges may form in a variant of BLC. A stabilising disulfide bridge may be constructed through the substitutions: S145C and T128C

Surface Charge Distribution

A variant with improved stability (typically improved thermostability or storage stability) as compared to the parent RP-II protease may be obtained by changing the surface charge distribution of the RP-II protease. For example, when the pH is lowered to about 5 or below, histidine residues typically become positively charged and, consequently, unfavorable electrostatic interactions on the protein surface may occur. By engineering the surface charge of the RP-II protease one may avoid such unfavorable electrostatic interactions that in turn may lead to a higher stability of the RP-II protease.

Charged amino acid residues are (a) positively charged: Lys, Arg, His (pH<5), Tyr (pH>9) and Cys (pH>10) and (b) negatively charged: Asp and Glu.

The surface charge distribution may be modified by (a) removing charged residues from the surface through deletion of a charged residue or substituting an uncharged residue for a charged residue, (b) adding charged residues to the surface through insertion of a charged residue or substituting a charged residue for an uncharged residue, or (c) by reverting the charge at a residue through substituting a positively charged residue for a negatively charged residue or substituting a negatively charged residue for a positively charged residue.

Therefore, a further aspect of the present invention relates to a method for constructing a variant of a parent RP-II protease having a modified surface charge distribution, the method comprising:

-   -   a) identifying, on the surface of the parent RP-II protease, at         least one charged amino acid residue;     -   b) modifying the charged residue identified in step (a) through         deletion or substitution with an uncharged amino acid residue;     -   c) optionally repeating steps a) and b) recursively;     -   d) preparing the variant resulting from steps a)-c);     -   e) testing the stability of said variant; and     -   f) optionally repeating steps a)-e) recursively; and     -   g) selecting a RP-II protease variant having increased stability         as compared to the parent RP-II protease.

As will be understood by the skilled person it may also, in some cases, be advantageous to substitute an uncharged amino acid residue with an amino acid residue bearing a charge or, alternatively, it may in some cases be advantageous to substitute an amino acid residue bearing a charge with an amino acid residue bearing a charge of opposite sign. Thus, the above-mentioned method may be employed by the skilled person also for these purposes. In the case of substituting an uncharged amino acid residue with an amino acid residue bearing a charge the above-mentioned method may be employed the only difference being steps a) and b) which will then read:

-   -   a) identifying, on the surface of the parent RP-II protease, at         least one position being occupied by an uncharged amino acid         residue;     -   b) modifying the charge in that position by substituting the         uncharged amino acid residue with a charged amino acid residue         or by insertion of a charged amino acid residue at the position.

Also in the case of changing the sign of an amino acid residue present on the surface of the RP-II protease the above method may be employed. Again, compared to the above method, the only difference being steps a) and b) which, in this case, read:

-   -   a) identifying, on the surface of the parent RP-II protease, at         least one charged amino acid residue;     -   b) substituting the charged amino acid residue identified in         step (a) with an amino acid residue having an opposite charge.

In order to determine the amino acid residues of a protease, which are present on the surface of the enzyme, the surface accessible area are measured using the DSSP program (Kabsch and Sander, Biopolymers (1983), 22, 2577-2637). All residues having a surface accessibility higher than 0, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55 or 0.60 are regarded a surface residue.

An amino acid residue found on the surface of BLC using the above method is T109 and it is contemplated that the substitutions T109R, K, H are of particular interest.

Similar substitutions may be introduced in equivalent positions of other RP-II proteases.

For the purpose of providing RP-II protease variants exhibiting improved wash performance it is possible to modify the pl of the RP-II protease through modification of the surface charge as indicated in WO 91/00345 (Novozymes A/S) and/or WO 99/20771 (Genencor International, Inc.)

Especially changing the pl of the RP-II protease is of interest

Changes in BLC: T109R, K, H Q143R, K, H E209Q, N D7N, S, T Q174R, K, H N216R, K, H Y17R, K, H Y95R, K, H

Corresponding modifications may be performed in corresponding positions of other RP-II proteases.

Substitution with Proline Residues

Improved thermostability of a RP-II protease can be obtained by subjecting the RP-II protease in question to analysis for secondary structure, identifying residues in the RP-II protease having dihedral angles φ (phi) and ψ (psi) confined to the intervals [−90°<φ<−40° and −180°<ψ−<180°], preferably the intervals [−90°<φ<−40° and 120°<ψ<180°] or [−90°<φ<−40° and −50°<ψ<10°] and excluding residues located in regions in which the RP-II protease is characterized by possessing α-helical or β-sheet structure.

After the dihedral angles φ (phi) and ψ (psi) for the amino acids have been calculated, based on the atomic structure in the crystalline RP-II proteases, it is possible to select position(s) which has/have dihedral phi and psi angles favourable for substitution with a proline residue. The aliphatic side chain of proline residues is bonded covalently to the nitrogen atom of the peptide group. The resulting cyclic five-membered ring consequently imposes a rigid constraint on the rotation about the N—C_(α) bond of the peptide backbone and simultaneously prevents the formation of hydrogen bonding to the backbone N-atom. For these structural reasons, proline residues are generally not compatible with α-helical and β-sheet secondary conformations.

If a proline residue is not already at the identified position(s), the naturally occurring amino acid residue is substituted with a proline residue, preferably by site directed mutagenesis applied on a gene encoding the RP-II protease in question.

In the group of BLC-like proteases proline residues can be introduced at positions 18, 115, 185, 269 and 293. Accordingly, a preferred BLC variant has one or more of the substitutions: T60P, S221P, G193P, and V194P.

Alteration of Activity:

Amino acid residues at a distance of less than 10 Å from the active site residues are most likely to influence the specificity and activity of the RP-II proteases, therefore variants comprising modifications in positions 1, 8, 22-35 (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35), 42-58 (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58), 82-100 (82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100), 129-135 (1129, 130, 131, 132, 133, 134, 135), 141-142, 153-156 (153, 154, 155, 156), 158, 161-171 (161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171), 188-193 (188, 189, 190, 191, 192, 193), 195, 201-207 (201, 202, 203, 204, 205, 206, 207), 210, 213-214, 217 may provide a change in activity and/or specificity of the RP-II protease variant.

Substrate Binding Site

The substrate binding site is identified by the residues in contact with a substrate model, such as the DAFE. The 3D structure coordinates of the BLC protease with DAFE bound in the active site can be found in Appendix 1. Without being limited to any theory, it is presently believed that binding between a substrate and an enzyme is supported by favorable interactions found within a sphere 10 Å from the substrate molecule, in particular within a sphere of 6 Å from the substrate molecule. Examples of such favorable bonds are hydrogen bonds, strong electrostatic interaction and/or hydrophobic interactions.

The following residues of the BLC protease (SEQ ID NO:1), are within a distance of 10 Å from the peptide DAFE and thus believed to be involved in interactions with said substrate: 1, 2, 3, 8, 25, 29, 30, 31, 32, 33, 34, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 90, 91, 92, 93, 94, 95, 96, 97, 129, 131, 132, 133, 134, 135, 155, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 171, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200 and 204.

The following residues of the BLC protease (SEQ ID NO: 1), are within a distance of 6 Å from the peptide DAFE and thus believed to be involved in interactions with said substrate: 1, 2, 31, 32, 47, 48, 88, 91, 93, 96, 162, 163, 164, 165, 166, 167, 168, 190, 191, 192, 193, 194, 195, and 201.

Helix Capping:

For the RP-II proteases helix capping may be obtained by modifying the position structurally corresponding to position 221 in BLC, and specifically in BLC by the modification A221N,T

Removal of Deamidation Sites

For the RP-II proteases, removal of deamidation sites may be obtained by modifying the positions structurally corresponding to positions 213, 216, and 222 of BLC, and specifically in BLC by the modifications.

N213A,C,D,E,F,G,H,I,K,L,P,Q,R,S,T,V,Y,M,W preferably N213L,T,S N216A,C,D,E,F,G,H,I,K,L,P,Q,R,S,T,V,Y,M,W preferably N216L,T,S N222A,C,D,E,F,G,H,I,K,L,P,Q,R,S,T,V,Y,M,W preferably N222L,T,S

Combined Modifications

The present invention also encompasses any of the above mentioned RP-II protease variants in combination with any other modification to the amino acid sequence thereof. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged. Such modifications to be combined with any of the above indicated modifications are exemplified in the following.

Removal of Critical Oxidation Sites

In order to increase the stability of the RP-II protease it may be advantageous to substitute or delete critical oxidation sites, such as methionines, with other amino acid residues which are not subject to oxidation.

Accordingly, in a further embodiment the present invention relates to an RP-II protease variant, in which one or more amino acid residues susceptible to oxidation, especially methionine residues exposed to the surface of the molecule, is/are deleted or replaced with another amino acid residue less susceptible to oxidation. The amino acid residue less susceptible to oxidation may for instance be selected from the group consisting of A, E, N, Q, I, L, S and K.

Specific such variants comprises at least one of the deletions or substitutions M36{*,S,A,N,Q,K}; M160{*,S,A,N,Q,K} of the BLC protease; M144{*,S,A,N,Q,K} of the AC116 and CDJ31 proteases; M67{*,S,A,N,Q,K}, M79{*,S,A,N,Q,K}, M137{*,S,A,N,Q,K}, M144{*,S,A,N,Q,K}, and M171{*,S,A,N,Q,K} of the BO32, BIP and JA96 proteases; M159{*,S,A,N,Q,K} of the BO32 protease; M81{*,S,A,N,Q,K}, and M141{*,S,A,N,Q,K} in the MPR protease; and M17{*,S,A,N,Q,K}, M67{*,S,A,N,Q,K}, M144{*,S,A,N,Q,K}, M160{*,S,A,N,Q,K}, M186{*,S,A,N,Q,K}, and M217{*,S,A,N,Q,K} of the AA513 protease (positions are indicated in relation to the BLC protease as indicated in FIG. 2).

Modification of Asn-Gly Sequences in the Protease

It is known that at alkaline pH, the side chain of Asn may interact with the NH group of a sequential neighboring amino acid to form an isoAsp residue where the backbone goes through the Asp side chain. This will leave the backbone more vulnerable to proteolysis. The deamidation is much more likely to occur if the residue that follows is a Gly. Changing the Asn in front of the Gly or the Gly will prevent this from happening and thus improve the stability, especially as concerns thermo- and storage stability.

The invention consequently further relates to an RP-II protease variant, in which either or both residues of any of the Asn-Gly sequence appearing in the amino acid sequence of the parent RP-II protease is/are deleted or substituted with a residue of a different amino acid.

The Asn and/or Gly residue may, for instance, be substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.

More specifically, any of the Asn or Gly residues of the Asn-Gly occupying positions 68-69, 182-183 and/or 192-193 of the BLC protease; positions 68-69 and/or 192-193 of the AC116 and CDJ-31 proteases, positions 45-46, 74-75, 196-197, and/or 201-202 of the BO32, JA96 and BIP proteases, positions 68-69, 103-104 and/or 192-196 of the MPR protease; and positions 90-91 and/or 201-202 of the AA513 protease, may be deleted or substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y. (positions are indicated in relation to the BLC protease as indicated in FIG. 2)

Specific Variants of BLC are: N68{*,A,Q,S,P,T,Y}; G69{*,A,Q,S,P,T,Y} N68{*,A,Q,S,P,T,Y}+G69{*,A,Q,S,P,T,Y} N182{*,A,Q,S,P,T,Y}; G183{*,A,Q,S,P,T,Y} N182{*,A,Q,S,P,T,Y}+G183{*,A,Q,S,P,T,Y} N192{*,A,Q,S,P,T,Y}; G193{*,A,Q,S,P,T,Y} N192{*,A,Q,S,P,T,Y}+G193{*,A,Q,S,P,T,Y}

and combinations thereof.

Specific Variants of the AC116 and CDJ-31 Proteases are: N68{*,A,Q,S,P,T,Y}; G69{*,A,Q,S,P,T,Y} N68{*,A,Q,S,P,T,Y}+G69{*,A,Q,S,P,T,Y} N192{*,A,Q,S,P,T,Y}; G193{*,A,Q,S,P,T,Y} N192{*,A,Q,S,P,T,Y}+G193{*,A,Q,S,P,T,Y} N68{*,A,Q,S,P,T,Y}+N192{*,A,Q,S,P,T,Y}

and combinations thereof.

Specific Variants of BO32, JA96 and BIP Proteases are: N45{*,A,Q,S,P,T,Y}; G46{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+G46{*,A,Q,S,P,T,Y} N74{*,A,Q,S,P,T,Y}; G75{*,A,Q,S,P,T,Y} N74{*,A,Q,S,P,T,Y}+G75{*,A,Q,S,P,T,Y} N196{*,A,Q,S,P,T,Y}; G197{*,A,Q,S,P,T,Y} N196{*,A,Q,S,P,T,Y}+G197{*,A,Q,S,P,T,Y} N201{*,A,Q,S,P,T,Y}; G202{*,A,Q,S,P,T,Y} N201{*,A,Q,S,P,T,Y}+G202{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+N74{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+N196{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y} N74{*,A,Q,S,P,T,Y}+N196{*,A,Q,S,P,T,Y} N74{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y} N196{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+N74{*,A,Q,S,P,T,Y}+N196{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+N74{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+N196{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y} N74{*,A,Q,S,P,T,Y}+N196{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y} N45{*,A,Q,S,P,T,Y}+N74{*,A,Q,S,P,T,Y}+N196{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y}

and combinations thereof.

Specific Variants of AA513 are: N90{*,A,Q,S,P,T,Y}; G91{*,A,Q,S,P,T,Y} N90{*,A,Q,S,P,T,Y}+G91{*,A,Q,S,P,T,Y} N201{*,A,Q,S,P,T,Y}; G202{*,A,Q,S,P,T,Y} N201{*,A,Q,S,P,T,Y}+G202{*,A,Q,S,P,T,Y} N90{*,A,Q,S,P,T,Y}+N201{*,A,Q,S,P,T,Y}

and combinations thereof.

Specific Variants of MPR are: N68{*,A,Q,S,P,T,Y}; G69{*,A,Q,S,P,T,Y} N68{*,A,Q,S,P,T,Y}+G69{*,A,Q,S,P,T,Y} N103{*,A,Q,S,P,T,Y}; G104{*,A,Q,S,P,T,Y} N103{*,A,Q,S,P,T,Y}+G104{*,A,Q,S,P,T,Y} N192{*,A,Q,S,P,T,Y}; G196{*,A,Q,S,P,T,Y} N192{*,A,Q,S,P,T,Y}+G196{*,A,Q,S,P,T,Y} N68{*,A,Q,S,P,T,Y}+N103{*,A,Q,S,P,T,Y} N68{*,A,Q,S,P,T,Y}+N192{*,A,Q,S,P,T,Y} N103{*,A,Q,S,P,T,Y}+N192{*,A,Q,S,P,T,Y} N68{*,A,Q,S,P,T,Y}+N103{*,A,Q,S,P,T,Y}+N192{*,A,Q,S,P,T,Y}

and combinations thereof.

Removal of Autoproteolysis Sites

According to a further aspect of the invention autoproteolysis sites may be removed by changing the amino acids at an autoproteolysis site. Since the RP-II proteases cleaves at Glu and Asp residues it is preferred to modify such residues of a parent RP-II protease having the same or a similar specificity, preferably by substituting with any other amino acid except Glu.

The parent RP-II proteases are mostly specific towards Glu and to a minor extent towards Asp residues. Therefore the modification of the parent (trypsin-like) RP-II protease may preferably be made by changing Glu to another amino acid residue (including Asp). Experiments have indicated that the substitution of Ala for Glu or Asp provides good results.

Glu and Asp residue are in the BLC, CDJ31 and AC116 proteases found in positions E101, E152, E173, E209, D6, D51, D96, D135, D161, and D212. BLC has a further Glu in position E104 and Asp in D7.

Specific BLC, CDJ31 and AC116 variants are thus E101A, E152A, E173A, E209A, D6A, D51A, D135A, D161A, D212A, and double, triple, quadruple, etc. combinations thereof. Further specific BLC variants are E104A and D7A.

In JA96, BO32 and BIP Glu and Asp are found at positions E81, E143, E151, E209, D5, D6, D69, D96, D103, D135, D152, D161, and D173.

Specific JA96, BO32 and BIP variants are thus E81A, E143A, E151A, E202A, D5A, D6A, D69A, D96A, D103A, D135A, D152A, D161A, D173A, and double, triple, quadruple, etc. combinations thereof.

In MPR Glu and Asp are found at positions E7, E89a, E152, D6, D54, D92, D96, D135, D144, D161, D177 and D209

Specific MPR variants are thus E7A, E89aA, E152A, D6A, D54A, D92A, D96A, D135A, D144A, D161A, D177A and D209A, and double, triple, quadruple, etc. combinations thereof.

In AA513 Glu and Asp are found at positions E26, E55, E94, E117, E123, E137b, E199, D40, D96, D103b, D103d, D135, D149, D154, D161, D184 and D209

Specific AA513 variants are thus E26A, E55A, E94A, E117A, E123A, E137bA, E199A, D40A, D96A, D103bA, D103dA, D135A, D149A, D154A, D161A, D184A and D209A, and double, triple, quadruple, etc. combinations thereof.

Corresponding variants are easily identified in any other RP-II protease.

Alternatively autoproteolysis can be prevented by changing the amino acid residue occupying the 1st and/or 2nd position following the Glu or Asp residue in question to Pro. For instance, this may in BLC, CDJ31 and AC116 be done in the positions 174 and/or 175 as follows:

Q174P; S175P; Q174P+5175P

or in a similar manner in JA96, BO32 or BIP at positions 152 and/or 153 as D152P; T153P; or D152P+T153P.

Corresponding variants are easily identified in these and any other RP-II protease.

Modification of Tryptophan Residues

In order to stabilize the protein it may be advantageous to replace or delete tryptophan residues at the surface of the protein, e.g., as described in U.S. Pat. No. 5,118,623. The tryptophan residues may advantageously be substituted for F, T, Q or G. Thus, in a further embodiment the invention relates to an RP-II variant comprising one or more of the following substitutions:

BLC and AC116: W35{F,T,Q,G}; W88{F,T,Q,G}; W142{F,T,Q,G}; W217{F,T,Q,G} CDJ31: W142{F,T,Q,G}; W217{F,T,Q,G}; BO32, JA96 and BIP: W142{F,T,Q,G}; AA513: W30{F,T,Q,G}; W72{F,T,Q,G}; W142{F,T,Q,G} MPR: W57{F,T,Q,G}; W88{F,T,Q,G}; W112{F,T,Q,G}; W142{F,T,Q,G}; W217{F,T,Q,G} Modification of Tyrosines

In relation to wash performance it has been found that the modification of certain tyrosine residues to phenylalanine provides an improved wash performance. Without being bound by any specific theory, it is believed that titration of these Tyr residues in the alkaline wash liquor has negative effects that are alleviated by replacing the Tyr residues with other residues, especially Phe or Trp, particularly Phe.

In the BLC, AC116 and CDJ31 parent RP-II proteases, the following tyrosine residues may be modified:

19, 50, 72, 74, 82, 95, 97, 112, 115, 117, 132, 154, 163, 195, 200. In BLC and CDJ31 the tyrosines in positions 17 and 158 may also be modified, and in AC116 and CDJ31 the tyrosines in position 172

Examples of specific variants comprise one or more of the following substitutions:

Y17{F,W}, Y19{F,W}, Y50{F,W}, Y72{F,W}, Y74{F,W}, Y82{F,W}, Y88{F,W}, Y95{F,W}, Y97{F,W}, Y112{F,W}, Y115{F,W}, Y117{F,W}, Y132{F,W}, Y154{F,W}, Y158{F,W}, Y163{F,W}, Y172{F,W}, Y195{F,W}, Y200{F,W}

In the JA96, BO32 and BIP parent RP-II proteases, the following tyrosine residues may be modified:

19, 24, 50, 57, 64, 83, 88, 95, 112, 132, 157, 158, 195, 216

Examples of specific JA96, BO32 and BIP variants comprises one or more of the following substitutions:

Y19{F,W}, Y24{F,W}, Y50{F,W}, Y57{F,W}, Y64{F,W}, Y83{F,W}, Y88{F,W}, Y95{F,W}, Y112{F,W}, Y132{F,W}, Y157{F,W}, Y158{F,W}, Y195{F,W} and Y216{F,W}

In the AA513 parent RP-II protease, the following tyrosine residues may be modified:

24, 74, 77, 84, 88, 97, 130, 132, 158, 163, 193a

Examples of specific AA513 variants comprises one or more of the following substitutions:

Y24{F,W}, Y74{F,W}, Y77{F,W}, Y84{F,W}, Y88{F,W}, Y97{F,W}, Y130{F,W}, Y132{F,W}, Y158{F,W}, Y163{F,W}, Y193A{F,W}

In the MPR parent RP-II protease, the following tyrosine residues may be modified:

19, 28a, 30, 50, 72, 74, 77, 83, 95, 97, 113, 115, 154, 158, 163, 172, 175, 200, 216

Examples of specific MPR variants comprises one or more of the following substitutions:

Y19{F,W}, Y28Ad{F,W}, Y30{F,W}, Y50{F,W}, Y72{F,W}, Y74{F,W}, Y77{F,W}, Y83{F,W}, Y95{F,W}, Y97{F,W}, Y113{F,W}, 115{F,W}, Y154{F,W}, Y158{F,W}, Y163{F,W}, Y172{F,W}, Y175{F,W}, Y200{F,W}, Y216{F,W}

Other Modifications for Combination

Examples of specific BLC variants comprises one or more of the following substitutions:

E152{A,R,K,G} E173A E209A E152G+G164R Methods of Preparing RP-II Protease Variants

The RP-II protease variants of the present invention may be produced by any known method within the art. The invention also relates to polynucleotides encoding the RP-II protease variants of the present invention, DNA constructs comprising such polynucleotides and host cells comprising such constructs or polynucleotides.

In general natural occurring proteins may be produced by culturing the organism expressing the protein and subsequently purifying the protein, or recombinantly by cloning a polynucleotide, e.g. genomic DNA or cDNA, encoding the protein into an expression vector, introducing said expression vector into a host cell, culturing the host cell and purifying the expressed protein.

Site-Directed Mutagenesis

Typically protein variants may be produced by site-directed mutagenesis of the gene encoding a parent protein, introduction of the mutated gene into an expression vector, host cell etc. The gene encoding the parent protein may be cloned from a strain producing the polypeptide or from an expression library, i.e. it may be isolated from genomic DNA or prepared from cDNA, or a combination thereof. The gene may even be a fully synthetically produced gene.

In general standard procedures for cloning of genes and/or introducing mutations (random and/or site directed) into said genes may be used in order to obtain a parent RP-II protease, or RP-II protease variant of the invention. For further description of suitable techniques reference is made to Molecular cloning: A laboratory manual (Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, N. Y.; Ausubel, F. M. et al. (eds.)); Current protocols in Molecular Biology (John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.)); Molecular Biological Methods for Bacillus (John Wiley and Sons, 1990); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); A Practical Guide To Molecular Cloning (B. Perbal, (1984)) and WO 96/34946.

Localized and Region Specific Random Mutagenesis

Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.

The random mutagenesis of a DNA sequence encoding a parent RP-II protease may be conveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present invention relates to a method for generating a variant of a parent RP-II protease wherein the variant exhibits an altered property, such as increased thermostability, increased stability at low pH and at low calcium concentration, relative to the parent RP-II protease, the method comprising:

-   -   a) subjecting a DNA sequence encoding the parent protease to         localized or region-specific random mutagenesis,     -   b) expressing the mutated DNA sequence obtained in step (a) in a         host cell, and     -   c) screening for host cells expressing a RP-II protease variant         which has an altered property relative to the parent RP-II         protease.

Step (a) of the above method of the invention is preferably performed using doped primers.

When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions that are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the RP-II protease by any published technique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, in which the percentage of wild-type and modification in each position is predefined. Furthermore, the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues. The doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% modifications in each position. An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints. The doping scheme may be made by using the DOPE program which, inter alia, ensures that introduction of stop codons is avoided (L. J. Jensen et al. Nucleic Acid Research, 26, 697-702 (1998).

The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent RP-II protease. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenizing agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.

In some cases it may be convenient to amplify the mutated DNA sequence prior to performing the expression step b) or the screening step c). Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenizing agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulants, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gram negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.

Localised Random Mutagenesis

The random mutagenesis may be advantageously localised to a part of the parent RP-II protease in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.

The localised or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above.

General Method for Localised Random Mutagenesis by Use of the DOPE Program

The localised random mutagenesis may be carried out by the following steps:

-   -   1. Select regions of interest for modification in the parent         enzyme     -   2. Decide on mutation sites and non-mutated sites in the         selected region     -   3. Decide on which kind of mutations should be carried out, e.g.         with respect to the desired stability and/or performance of the         variant to be constructed     -   4. Select structurally based mutations     -   5. Adjust the residues selected in step 3 with regard to step 4.     -   6. Analyse by use of a suitable dope algorithm the nucleotide         distribution.     -   7. If necessary, adjust the wanted residues to genetic code         realism, e.g. taking into account constraints resulting from the         genetic code, e.g. in order to avoid introduction of stop         codons; the skilled person will be aware that some codon         combinations cannot be used in practice and will need to be         adapted     -   8. Make primers     -   9. Perform localised random mutagenesis by use of the primers     -   10. Select resulting RP-II protease variants by screening for         the desired improved properties.

Suitable dope algorithms for use in step 6 are well known in the art. One such algorithm is described by Tomandl, D. et al., 1997, Journal of Computer-Aided Molecular Design 11:29-38. Another algorithm is DOPE (Jensen, L J, Andersen, K V, Svendsen, A, and Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).

Expression Vectors

A recombinant expression vector comprising a nucleic acid sequence encoding a RP-II protease variant of the invention may be any vector that may conveniently be subjected to recombinant DNA procedures and which may bring about the expression of the nucleic acid sequence.

The choice of vector will often depend on the host cell into which it is to be introduced. Examples of a suitable vector include a linear or closed circular plasmid or a virus. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMβ1. Examples of origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).

Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Vectors which are integrated into the genome of the host cell may contain any nucleic acid sequence enabling integration into the genome; in particular it may contain nucleic acid sequences facilitating integration into the genome by homologous or non-homologous re-combination. The vector system may be a single vector, e.g. plasmid or virus, or two or more vectors, e.g. plasmids or virus', which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

The vector may in particular be an expression vector in which the DNA sequence encoding the RP-II protease variant of the invention is operably linked to additional segments or control sequences required for transcription of the DNA. The term, “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence encoding the RP-II protease variant. Additional segments or control sequences include a promoter, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator. At a minimum the control sequences include a promoter and transcriptional and translational stop signals.

The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus subtilis levansucrase gene (sacB), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731). Other examples include the phage Lambda P_(R) or P_(L) promoters or the E. coli lac, trp or tac promoters or the Streptomyces coelicolor agarase gene (dagA). Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S. Pat. No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral (-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters. Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter.

Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4-c (Russell et al., Nature 304 (1983), 652-654) promoters.

Further useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488. In a mammalian host cell, useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).

Examples of suitable promoters for use in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814) or the adenovirus 2 major late promoter.

An example of a suitable promoter for use in insect cells is the polyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

The DNA sequence encoding a RP-II protease variant of the invention may also, if necessary, be operably connected to a suitable terminator.

The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, resistance. A frequently used mammalian marker is the dihydrofolate reductase gene (DHFR). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species. Particularly, for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.

To direct a RP-II protease variant of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al.).

More than one copy of a nucleic acid sequence encoding an enzyme of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.

The nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention. The nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.

Host Cells

The DNA sequence encoding a RP-II protease variant of the present invention may be either homologous or heterologous to the host cell into which it is introduced. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term “homologous” is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term “heterologous” is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell that is capable of producing the present RP-II protease variants, such as prokaryotes, e.g. bacteria or eukaryotes, such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells.

Examples of bacterial host cells which, on cultivation, are capable of producing the RP-II protease variants of the invention are gram-positive bacteria such as strains of Bacillus, e.g. strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram-negative bacteria such as Escherichia coli or Pseudomonas sp.

The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).

When expressing the RP-II protease variant in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.

When expressing the RP-II protease variant in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or it may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.

Examples of host yeast cells include cells of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansehula, or Yarrowia. In a particular embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Other useful yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansehula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279 and U.S. Pat. No. 4,879,231). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B. J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose, A. H., and Harrison, J. S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981). Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920.

Examples of filamentous fungal cells include filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra), in particular it may of the a cell of a species of Acremonium, such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A. oryzae, Fusarium, such as F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. heterosporum, F. negundi, F. reticulatum, F. roseum, F. sambucinum, F. sarcochroum, F. sulphureum, F. trichothecioides or F. oxysporum, Humicola, such as H. insolens or H. lanuginose, Mucor, such as M. miehei, Myceliophthora, such as M. thermophilum, Neurospora, such as N. crassa, Penicillium, such as P. purpurogenum, Thielavia, such as T. terrestris, Tolypocladium, or Trichoderma, such as T. harzianum, T. koningii, T. longibrachiatum, T. reesei or T. viride, or a teleomorph or synonym thereof. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.

Examples of insect cells include a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214). Culture conditions may suitably be as described in WO 89/01029 or WO 89/01028. Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in U.S. Pat. No. 4,745,051; U.S. Pat. No. 4,775,624; U.S. Pat. No. 4,879,236; U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222; EP 397,485).

Examples of mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N. Y., 1987, Hawley-Nelson et al., Focus 15 (1993), 73; Ciccarone et al., Focus 15 (1993), 80; Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845. Mammalian cells may be transfected by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).

Methods for Expression and Isolation of Proteins

To express an enzyme of the present invention the above mentioned host cells trans-formed or transfected with a vector comprising a nucleic acid sequence encoding an enzyme of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.

The medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).

If the enzymes of the present invention are secreted into the nutrient medium, they may be recovered directly from the medium. If they are not secreted, they may be recovered from cell lysates. The enzymes of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the enzyme in question.

The enzymes of the invention may be detected using methods known in the art that are specific for these proteins. These detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activity are known in the art.

The enzymes of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

When an expression vector comprising a DNA sequence encoding an enzyme of the present invention is transformed/transfected into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme. An advantage of using a heterologous host cell is that it is possible to make a highly purified enzyme composition, characterized in being free from homologous impurities, which are often present when a protein or peptide is expressed in a homologous host cell. In this context homologous impurities mean any impurity (e.g. other polypeptides than the enzyme of the invention) which originates from the homologous cell where the enzyme of the invention is originally obtained from.

Detergent Applications

The enzyme of the invention may be added to and thus become a component of a detergent composition.

The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Proteases:

Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 68, 76, 87, 97, 101, 104, 106, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, 245, 252 and 274.

Preferred commercially available protease enzymes include Alcalase™, Savinase™ Primase™, Duralase™, Esperase™, Coronase™ and Kannase™ (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include Lipolase™, Lipolase Ultra™ and Lipex™ (Novozymes A/S).

Amylases:

Suitable amylases (α and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Stainzyme™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International Inc.).

Cellulases:

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Renozyme™, Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.

When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂ source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzyme protein per litre of wash liquor, in particular 0.1-1 mg of enzyme protein per litre of wash liquor.

The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.

Food Processing Applications

The RP-II protease variants of the present invention may also be used in the processing of food, especially in the field of diary products, such as milk, cream and cheese, but also in the processing of meat and vegetables.

Feed Processing Application

The RP-II protease variants of the present invention may also be used in the processing of feed for cattle, poultry, and pigs and especially for pet food.

Treatment of Hides

The RP-II protease variants of the invention may also be used for the treatment of hides.

Decontamination of Possibly Infested Materials

The RP-II protease variants of the invention may also be used in processes for decontaminating instruments, surfaces, and other materials in hospitals, clinics, and meat processing plants, etc. in order to decompose prions or other infectious agents.

Materials and Methods Strains:

B. subtilis DN1885: Disclosed in WO 01/16285

Plasmids:

pNM1003: Disclosed in WO 01/16285 pSX222: Disclosed in WO 96/34946 pNM1008: See Example 2

Method for Producing a Protease Variant

The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.

When an expression vector comprising a DNA sequence encoding the enzyme is trans-formed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention. Thereby it is possible to make a highly purified RP-II protease composition, characterized in being free from homologous impurities.

The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed RP-II protease may conveniently be secreted into the culture medium and may be recovered there-from by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Proteolytic Activity

Enzyme activity can be measured using the PNA assay using succinyl-alanine-alanine-proline-glutamicacid-paranitroaniline as a substrate. The principle of the PNA assay is described in the Journal of American Oil Chemists Society, Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).

Textiles

Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland. Especially type EMPA 116 (cotton textile stained with blood, milk and ink) and EMPA 117 (polyester/cotton textile stained with blood, milk and ink). The textile can be cut into a smaller textile piece of 5×3 cm or 13×3 cm

Other relevant protease stain may be used as well, e.g. C-03, C-05, C-10 from CFT, Center For Testmaterials, Vlaardingen, Netherlands

Wash Conditions

Latin North Region America Europe America Japan Temperature 20° C. 30° C. 20° C. 20° C. Washing time 14 min 20 min 12 min 15 min Swatches EMPA 117 EMPA 116 EMPA 117 EMPA 117 Water 9 or 12° dH 15° dH 6° dH 3° dH Hardness* Detergent 1.5 or 2.5 g/l 4, 6 or 8 g/l HDL: 1.5 g/l 0.5 or 0.7 dosage Washing pH As is, or As is, or As is, or As is, or adjusted adjusted adjusted adjusted to 8, 9, 10 to 8, 9, 10 to 8, 9, 10 to 8, 9, 10 *° dH: adjusted by adding CaCl₂*2H₂O; MgCl₂*6H₂O; NaHCO₃ (Ratio Ca²⁺:Mg²⁺:HCO³⁻ = 2:1:6) to milli-Q water.

Detergents

The enzymes of the invention may be tested in the detergent formulations disclosed in WO 97/07202 or in detergents formulations purchased from wfk testgewebe GmbH or similar supplier

List of test detergents from wfk testgewebe

-   -   IEC 60456 Type A* Base Detergent     -   IEC 60456 Type B Base Detergent     -   IEC 60456 Type C Detergent     -   ECE Reference Detergent with Phosphate (1977)     -   ECE Reference Detergent without Phosphate (1998)     -   AHAM Standard Detergent     -   EU ECOLABEL (detergents) Light Duty Detergent     -   EU ECOLABEL (detergents) PVP         However, also one of the following commercial detergents may be         used in the wash assay, e.g.

Omo Multi Acao HDP, Unilever, Brazil

Tide HDL, P&G, US

Wisk HDL, Unilever, US

TOP HDP, Lion, Japan

Attack HDP, Kao, Japan

Ariel Regular HDP, P&G, Europe

Ariel Compact HDPC, P&G, Europe

Persil Megaperls, Henkel, Germany

Persil, Unilever, UK

Furthermore, a brand extension or color/compact version for the above specified detergent could be used as well If the detergent contains enzymes, the detergent should be in-activated before use in order to eliminate the enzyme activity already present in the detergent. This is done by heating a detergent stock solution to 85° C. in 5 minutes in a micro wave oven. The concentration of the detergent stock solution in the micro wave oven is between 4-20 g/l

Example 1 Modelling RP-II Proteases from the 3D Structure of BLC

The overall homology of Bacillus licheniformis protease BCL to other RP-II proteases is high. The similarity between the different RP-II proteases is provided in Table 1. Using the sequence alignment of FIG. 2 a model of the JA96 protease can be build using a suitable modelling tool like the Accellrys software Homology, or Modeller (also from Accellrys), or other software like Nest. These programs provide results as a first rough model, with some optimization in the Modeller and Nest programs.

The first rough model provides a close structural homology between the model of JA96 protease and the 3D structure of the BCL as there are no overlapping side chains in the model structure. To optimize the structure the protein can in silico be soaked in a box of water and subjected to energy minimization and further molecular dynamics simulations using e.g. the CHARMm™ software from Accelrys. The in silico soaking in water can conveniently be done by adding water in the Insight II program (from Accelrys) with a box size of 75*75*75 Å³. The energy minimization can be done using settings of 300 Steepest descent (SD) and further 600 Conjugated gradients (CJ). The molecular dynamics simulations can conveniently be done using 1.2 ns run using the Verlet algorithm at 300K and standard parameters (see CHARMm manual). Other RP-II protease 3D models may be built in an analogous way.

Example 2 Construction of Library of RP-II Protease Variants Construction and Expression of BLC

A B. subtilis-E. coli shuttle vector, pNM1003, suited to a gene coding for RP-II protease BLC and its mutants was constructed. It is derived from the B. subtilis expression vector pSX222 (Described in WO 96/34946) as described in WO 01/16285. To facilitate cloning pNM1008 was constructed introducing a kpnI restriction site downstream the HindIII site to facilitate the cloning of fragments inside the vector. For transformation in Bacillus pNM1008 was restricted with HindIII and a 4350 by DNA fragment was isolated and ligated. The ligation mixture was used to transform competent B. subtilis DN1885, selecting for protease activity, as described in WO 01/16285.

Site-Directed Mutagenesis

BLC site-directed variants of the invention comprising specific substitutions, insertions or deletions in the molecule are made by traditional cloning of PCR fragments (Sambrook et. al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor) produced by oligonucleotides containing the desired modification. As template pNM1008 is used. In a first PCR using a mutational primer (anti-sense) with a suitable opposite sense primer (e.g. 5″-CTGTGCCCTTTAACCGCACAGC (SEQ ID No. 17)), downstream of the MluI site is used. The resulting DNA fragment is used as a sense primer in a second PCR together with a suitable anti-sense primer (e.g. 5′-GCATAAGCTTTTACAGGTACCGGC (SEQ ID No. 18)) upstream from the KpnI digestion site. This resulting PCR product is digested with KpnI and MluI and ligated in pNM1008 digested with the respective enzymes.

The ligation reaction is transformed into E. coli by well-known techniques and 5 randomly chosen colonies are sequenced to confirm the designed mutations.

In order to express a BLC variant of the invention, the pNM1008 derived plasmid comprising the variant is digested with HindIII, ligated and transformed into a competent B. subtilis strain, selecting for protease activity.

Example 3 Purification of Enzymes and Variants

This procedure relates to purification of 2 liter scale fermentation for the production of the RP-II proteases of the invention in a Bacillus host cell.

Approximately 1.6 liters of fermentation broth are centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The supernatants are adjusted to pH 7 using 10% acetic acid and filtered through a Seitz Supra S100 filter plate.

At room temperature, the filtrate is applied to a 100 ml Bacitracin affinity column equilibrated with 0.01M dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7 with sodium hydroxide (Buffer A). After washing the column with Buffer A to remove unbound protein, the protease is eluted from the Bacitracin column using Buffer A supplemented with 25% 2-propanol and 1 M sodium chloride.

The fractions with protease activity from the Bacitracin purification step are combined and applied to a 750 ml Sephadex G25 column (5 cm dia.) equilibrated with Buffer A.

Fractions with proteolytic activity from the Sephadex G25 column are combined and the pH was adjusted to pH 6 with 10% acetic acid and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm dia.) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.1 M boric acid, and 0.002 M calcium chloride adjusted to pH 6 with sodium hydroxide.

The protease is eluted using a linear gradient of 0-0.2 M sodium chloride in 2 liters of the same buffer.

Finally, the protease containing fractions from the CM Sepharose column are combined and filtered through a 0.2μ filter.

By using the techniques of Example 2 for the construction of variants and fermentation, and the above isolation procedure the following RP-II proteases and variants thereof may be produced and isolated:

Example 4 Wash Performance of Detergent Compositions Comprising Modified Enzymes AMSA

The enzyme variants of the present application is tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress. For further description see WO 02/42740 especially the paragraph “Special method embodiments” at page 23-24.

The assay is conducted under the experimental conditions specified below. In respect of the detergent used, all the detergents listed above under “Materials and Methods” may be used:

Detergent base Example: Omo Acao Detergent dosage Example: 1.5 g/l Test solution volume 160 micro l pH Example: As is Wash time Example: 14 minutes Temperature Example: 20° C. Water hardness Example: 9° dH Enzyme concentration in test solution 5 nM, 10 nM and 30 nM Test material Example: EMPA 117

After washing the textile pieces is flushed in tap water and air-dried.

The performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific enzyme variant. Brightness can also be expressed as the intensity of the light reflected from the textile sample when luminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore the intensity of the reflected light can be used to measure wash performance of an enzyme variant.

Colour measurements are made with a professional flatbed scanner (PFU DL2400pro), which is used to capture an image of the washed textile samples. The scans are made with a resolution of 200 dpi and with an output colour dept of 24 bits. In order to get accurate results, the scanner is frequently calibrated with a Kodak reflective IT8 target.

To extract a value for the light intensity from the scanned images, a special designed software application is used (Novozymes Color Vector Analyzer). The program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:

Int=√{square root over (r² +g ² +b ²)}.

The wash performance (P) of the variants is calculated in accordance with the below formula:

P=Int(v)−Int(r)

where Int(v) is the light intensity value of textile surface washed with enzyme variant and Int(r) is the light intensity value of textile surface washed with the reference enzyme BLC.

A performance score is given as the result of the miniwash in accordance with the definition:

Performance Scores (S) are summing up the performances (P) of the tested enzyme variants as:

S=2 which indicates that the variant performs better than the reference at all three concentrations (5, 10 and 30 nM) and S=1 which indicates that the variant performs better than the reference at one or two concentrations.

A variant is considered to exhibit improved wash performance, if it performs better than the reference in at least one detergent composition.

Mini Wash Assay

The millilitre scale wash performance assay is conducted under the following conditions:

Detergent base Example: Omo Acao detergent powder Detergent dose Example: 1.5 g/l pH Example: “as is” in the current detergent solution and is not adjusted. Wash time Example: 14 min. Temperature Example: 20° C. Water hardness Example: 9° dH, adjusted by adding CaCl₂*2H₂O; MgCl₂*6H₂O; NaHCO₃ (Ca²⁺:Mg²⁺:HCO³⁻ = 2:1:6) to milli-Q water. Enzymes Variants of BLC. BLC as reference enzyme Enzyme conc. 5 nM, 10 nM, 30 nM Test system 125 ml glass beakers. Textile dipped in test solution. Continuously lifted up and down into the detergent solution, 50 times per minute (up-time 0.4 sec, down- time 0.4 sec, lift time 0.2 sec) Test solution 50 ml volume Test material Example: EMPA 117 textile swatches (13 × 5 cm)

After washing the textile piece is flushed in tap water and air-dried and the remission from the test material is measured at 460 nm using a Zeiss MCS 521 VIS spectrophotometer. The measurements are done according to the manufacturer's protocol.

A performance score is given as the result of the miniwash in accordance with the definition:

Performance Scores (S) are summing up the performances (P) of the tested enzyme variants as:

S=2 which indicates that the variant performs better than the reference at all three concentrations (5, 10 and 30 nM) and S=1 which indicates that the variant performs better than the reference at one or two concentrations.

A Performance Score higher than 1 indicates better wash performance.

A variant is considered to exhibit improved wash performance, if it performs better than the reference in at least one detergent composition.

The following RP-II variants were constructed as indicated in Example 2 to be purified in accordance with Example 3 and tested as indicated above:

Ion-Binding Modification: D7E; D7Q; H144R; D161R; D161K; H144Q+D161R Mobility Modification: G30A; G91A Cys-Bridge Formation: S145C+T128C Surface Charge Modification: D7N,S,T; Y17R,K,H; Y95R,K,H; T109R,K,H; Q143R,K,H; Q174R,K,H; E209Q,N; N216R,K,H Proline Stability: T60P; S221 P; G193P; V194P Example 5 Storage Stability of Modified Enzymes

The storage stability of the variants of the invention is determined by measuring the “residual activity” of the parent and the variants at regular time intervals. The storage stability is often expressed as the half-life, T_(1/2), the time lapsed till the activity is half the initial value.

Residual activity=(Activity at t=i)/((Activity at t=0)×100)%

The Proteolytic activity is measured as described above (PNA assay).

Example 6 Thermostability of Modified Enzymes

The thermostability of the protease variant s of the invention is determine by Differential Scanning Calorimetry (DSC) typically with a heating rate of 0.5° C. per minute in a solution containg about 2 mg/ml variant.

Example 7 Autoproteolytic Stability Of Modified Enzymes Comparative Fermentation Experiment

The RP-II variants of the invention are in a fermentation experiment compared to the parent RP-II protease.

Both the variants and the parent are cloned in a pNM1008 expression vector background and fermented in a suitable medium.

After 5 days fermentation 1.5 ml of the fermentation medium is centrifuged and the supernatant used to measure the Proteolytic activity (KPNU) as described above.

The variants providing an increased proteolytic activity in comparison to the activity of the parent are considered to posses an improved autoproteolytic stability relative to the parent.

Example 8 Oxidation Stability of Modified Enzymes

The variants are tested for their oxidation stability in 0.01 M peracetic acid after 20 minutes at 50° C. and pH 7. The parent protease is used as reference.

The results are presented by the residual proteolytic activity in the heat treated samples relative to samples untreated by oxidant or heat.

APPENDIX 1 ATOM 3359 N SER B 1 −2.987 12.370 17.565 1.00 7.82 N ATOM 3361 CA SER B 1 −2.255 12.820 16.353 1.00 7.97 C ATOM 3363 CB SER B 1 −3.233 12.933 15.188 1.00 8.69 C ATOM 3366 OG SER B 1 −3.995 11.748 15.028 1.00 9.01 O ATOM 3368 C SER B 1 −1.637 14.171 16.602 1.00 8.14 C ATOM 3369 O SER B 1 −2.098 14.938 17.439 1.00 8.05 O ATOM 3372 N VAL B 2 −0.592 14.472 15.848 1.00 8.60 N ATOM 3374 CA VAL B 2 −0.039 15.812 15.824 1.00 10.11 C ATOM 3376 CB VAL B 2 1.432 15.811 15.404 1.00 11.81 C ATOM 3378 CG1 VAL B 2 1.949 17.239 15.233 1.00 13.46 C ATOM 3382 CG2 VAL B 2 2.255 15.065 16.421 1.00 14.12 C ATOM 3386 C VAL B 2 −0.867 16.605 14.830 1.00 10.56 C ATOM 3387 O VAL B 2 −0.928 16.250 13.660 1.00 12.81 O ATOM 3388 N ILE B 3 −1.524 17.640 15.331 1.00 9.91 N ATOM 3390 CA ILE B 3 −2.409 18.487 14.537 1.00 10.49 C ATOM 3392 CB ILE B 3 −3.747 18.700 15.279 1.00 10.68 C ATOM 3394 CG1 ILE B 3 −4.452 17.348 15.457 1.00 10.36 C ATOM 3397 CD1 ILE B 3 −5.671 17.398 16.350 1.00 11.17 C ATOM 3401 CG2 ILE B 3 −4.638 19.704 14.531 1.00 13.34 C ATOM 3405 C ILE B 3 −1.683 19.796 14.299 1.00 10.96 C ATOM 3406 O ILE B 3 −1.332 20.502 15.234 1.00 10.91 O ATOM 3407 N GLY B 4 −1.433 20.141 13.043 1.00 12.22 N ATOM 3409 CA GLY B 4 −0.702 21.359 12.748 1.00 12.69 C ATOM 3412 C GLY B 4 0.685 21.285 13.344 1.00 12.61 C ATOM 3413 O GLY B 4 1.324 20.239 13.303 1.00 13.40 O ATOM 3414 N SER B 5 1.162 22.383 13.913 1.00 11.93 N ATOM 3416 CA SER B 5 2.466 22.358 14.557 1.00 11.64 C ATOM 3418 CB SER B 5 2.900 23.757 14.975 1.00 11.92 C ATOM 3421 OG SER B 5 2.011 24.329 15.906 1.00 13.28 O ATOM 3423 C SER B 5 2.438 21.451 15.770 1.00 11.22 C ATOM 3424 O SER B 5 1.437 21.366 16.462 1.00 11.19 O ATOM 3425 N ASP B 6 3.551 20.779 16.028 1.00 10.41 N ATOM 3427 CA ASP B 6 3.704 19.951 17.230 1.00 10.02 C ATOM 3429 CB ASP B 6 4.700 18.839 16.981 1.00 10.75 C ATOM 3432 CG ASP B 6 4.838 17.886 18.144 1.00 10.38 C ATOM 3433 OD1 ASP B 6 4.132 18.013 19.178 1.00 10.80 O ATOM 3434 OD2 ASP B 6 5.685 16.961 18.055 1.00 11.46 O ATOM 3435 C ASP B 6 4.185 20.807 18.373 1.00 9.61 C ATOM 3436 O ASP B 6 5.353 21.229 18.410 1.00 11.09 O ATOM 3437 N ASP B 7 3.290 21.057 19.312 1.00 8.85 N ATOM 3439 CA ASP B 7 3.582 21.969 20.387 1.00 8.21 C ATOM 3441 CB ASP B 7 2.453 23.010 20.550 1.00 9.26 C ATOM 3444 CG ASP B 7 2.334 23.975 19.386 1.00 10.17 C ATOM 3445 OD1 ASP B 7 3.147 23.902 18.444 1.00 11.15 O ATOM 3446 OD2 ASP B 7 1.377 24.778 19.332 1.00 10.99 O ATOM 3447 C ASP B 7 3.856 21.237 21.712 1.00 8.24 C ATOM 3448 O ASP B 7 3.978 21.870 22.753 1.00 8.50 O ATOM 3449 N ARG B 8 4.016 19.918 21.677 1.00 7.90 N ATOM 3451 CA ARG B 8 4.429 19.187 22.872 1.00 7.81 C ATOM 3453 CB ARG B 8 4.444 17.681 22.634 1.00 7.75 C ATOM 3456 CG ARG B 8 3.068 17.077 22.470 1.00 7.65 C ATOM 3459 CD ARG B 8 3.090 15.631 22.015 1.00 7.89 C ATOM 3462 NE ARG B 8 3.673 15.554 20.679 1.00 8.24 N ATOM 3464 CZ ARG B 8 4.023 14.422 20.073 1.00 8.49 C ATOM 3465 NH1 ARG B 8 3.781 13.244 20.628 1.00 8.61 N ATOM 3468 NH2 ARG B 8 4.622 14.472 18.909 1.00 9.63 N ATOM 3471 C ARG B 8 5.812 19.628 23.321 1.00 8.24 C ATOM 3472 O ARG B 8 6.684 19.907 22.505 1.00 9.34 O ATOM 3473 N THR B 9 6.007 19.640 24.632 1.00 8.26 N ATOM 3475 CA THR B 9 7.315 19.897 25.226 1.00 8.75 C ATOM 3477 CB THR B 9 7.368 21.243 25.939 1.00 9.87 C ATOM 3479 OG1 THR B 9 6.296 21.350 26.880 1.00 10.91 O ATOM 3481 CG2 THR B 9 7.191 22.375 24.936 1.00 11.78 C ATOM 3485 C THR B 9 7.660 18.787 26.199 1.00 8.34 C ATOM 3486 O THR B 9 6.793 18.176 26.835 1.00 8.22 O ATOM 3487 N ARG B 10 8.954 18.535 26.340 1.00 8.65 N ATOM 3489 CA ARG B 10 9.413 17.459 27.194 1.00 8.98 C ATOM 3491 CB ARG B 10 10.873 17.096 26.927 1.00 10.45 C ATOM 3494 CG ARG B 10 11.309 15.787 27.587 1.00 11.25 C ATOM 3497 CD ARG B 10 12.701 15.396 27.212 1.00 12.23 C ATOM 3500 NE ARG B 10 13.213 14.299 28.025 1.00 12.62 N ATOM 3502 CZ ARG B 10 14.465 13.868 27.967 1.00 14.40 C ATOM 3503 NH1 ARG B 10 15.328 14.413 27.114 1.00 16.93 N ATOM 3506 NH2 ARG B 10 14.855 12.884 28.743 1.00 14.13 N ATOM 3509 C ARG B 10 9.237 17.885 28.642 1.00 8.65 C ATOM 3510 O ARG B 10 9.534 19.027 29.025 1.00 9.59 O ATOM 3511 N VAL B 11 8.771 16.952 29.453 1.00 8.69 N ATOM 3513 CA VAL B 11 8.751 17.118 30.893 1.00 9.52 C ATOM 3515 CB VAL B 11 7.810 16.080 31.532 1.00 9.21 C ATOM 3517 CG1 VAL B 11 7.862 16.145 33.047 1.00 10.41 C ATOM 3521 CG2 VAL B 11 6.381 16.257 31.015 1.00 9.54 C ATOM 3525 C VAL B 11 10.207 16.954 31.390 1.00 10.62 C ATOM 3526 O VAL B 11 10.777 15.869 31.301 1.00 12.34 O ATOM 3527 N THR B 12 10.795 18.048 31.884 1.00 12.38 N ATOM 3529 CA THR B 12 12.217 18.113 32.253 1.00 13.55 C ATOM 3531 CB THR B 12 12.790 19.543 32.093 1.00 14.37 C ATOM 3533 OG1 THR B 12 12.035 20.449 32.902 1.00 17.60 O ATOM 3535 CG2 THR B 12 12.611 20.030 30.671 1.00 16.03 C ATOM 3539 C THR B 12 12.507 17.657 33.666 1.00 13.34 C ATOM 3540 O THR B 12 13.669 17.515 34.032 1.00 14.60 O ATOM 3541 N ASN B 13 11.472 17.465 34.469 1.00 12.04 N ATOM 3543 CA ASN B 13 11.646 16.901 35.800 1.00 11.12 C ATOM 3545 CB ASN B 13 11.713 17.962 36.894 1.00 11.74 C ATOM 3548 CG ASN B 13 11.935 17.344 38.252 1.00 12.29 C ATOM 3549 OD1 ASN B 13 12.166 16.141 38.356 1.00 12.18 O ATOM 3550 ND2 ASN B 13 11.868 18.153 39.302 1.00 15.45 N ATOM 3553 C ASN B 13 10.502 15.940 36.074 1.00 10.21 C ATOM 3554 O ASN B 13 9.450 16.321 36.578 1.00 10.60 O ATOM 3555 N THR B 14 10.714 14.678 35.743 1.00 9.43 N ATOM 3557 CA THR B 14 9.671 13.680 35.934 1.00 9.11 C ATOM 3559 CB THR B 14 9.887 12.455 35.046 1.00 9.24 C ATOM 3561 OG1 THR B 14 11.122 11.827 35.409 1.00 9.63 O ATOM 3563 CG2 THR B 14 9.958 12.808 33.561 1.00 10.29 C ATOM 3567 C THR B 14 9.556 13.227 37.385 1.00 9.62 C ATOM 3568 O THR B 14 8.730 12.361 37.672 1.00 10.68 O ATOM 3569 N THR B 15 10.357 13.804 38.295 1.00 10.09 N ATOM 3571 CA THR B 15 10.147 13.593 39.725 1.00 10.57 C ATOM 3573 CB THR B 15 11.456 13.495 40.553 1.00 11.89 C ATOM 3575 OG1 THR B 15 12.124 14.763 40.616 1.00 12.96 O ATOM 3577 CG2 THR B 15 12.432 12.491 39.954 1.00 12.96 C ATOM 3581 C THR B 15 9.244 14.638 40.367 1.00 10.41 C ATOM 3582 O THR B 15 8.911 14.514 41.540 1.00 12.03 O ATOM 3583 N ALA B 16 8.832 15.656 39.622 1.00 10.32 N ATOM 3585 CA ALA B 16 7.900 16.643 40.148 1.00 10.73 C ATOM 3587 CB ALA B 16 7.927 17.897 39.301 1.00 11.48 C ATOM 3591 C ALA B 16 6.488 16.060 40.161 1.00 10.05 C ATOM 3592 O ALA B 16 6.059 15.433 39.198 1.00 9.80 O ATOM 3593 N TYR B 17 5.755 16.284 41.237 1.00 10.35 N ATOM 3595 CA TYR B 17 4.338 15.962 41.260 1.00 10.36 C ATOM 3597 CB TYR B 17 3.838 16.018 42.706 1.00 10.90 C ATOM 3600 CG TYR B 17 2.379 15.675 42.858 1.00 10.77 C ATOM 3601 CD1 TYR B 17 1.436 16.674 42.985 1.00 11.41 C ATOM 3603 CE1 TYR B 17 0.086 16.386 43.118 1.00 11.35 C ATOM 3605 CZ TYR B 17 −0.338 15.081 43.139 1.00 11.51 C ATOM 3606 OH TYR B 17 −1.690 14.831 43.268 1.00 13.22 O ATOM 3608 CE2 TYR B 17 0.579 14.051 42.988 1.00 11.13 C ATOM 3610 CD2 TYR B 17 1.940 14.358 42.861 1.00 11.24 C ATOM 3612 C TYR B 17 3.588 16.946 40.363 1.00 10.06 C ATOM 3613 O TYR B 17 3.857 18.150 40.452 1.00 11.57 O ATOM 3614 N PRO B 18 2.609 16.510 39.557 1.00 10.05 N ATOM 3615 CA PRO B 18 2.080 15.145 39.436 1.00 9.55 C ATOM 3617 CB PRO B 18 0.606 15.412 39.151 1.00 10.69 C ATOM 3620 CG PRO B 18 0.646 16.604 38.275 1.00 11.31 C ATOM 3623 CD PRO B 18 1.772 17.460 38.810 1.00 10.99 C ATOM 3626 C PRO B 18 2.667 14.326 38.287 1.00 8.62 C ATOM 3627 O PRO B 18 2.189 13.217 38.035 1.00 8.43 O ATOM 3628 N TYR B 19 3.695 14.844 37.616 1.00 8.36 N ATOM 3630 CA TYR B 19 4.343 14.126 36.531 1.00 8.21 C ATOM 3632 CB TYR B 19 5.389 15.034 35.875 1.00 8.56 C ATOM 3635 CG TYR B 19 4.722 16.277 35.304 1.00 8.70 C ATOM 3636 CD1 TYR B 19 4.072 16.231 34.070 1.00 8.24 C ATOM 3638 CE1 TYR B 19 3.424 17.343 33.553 1.00 9.10 C ATOM 3640 CZ TYR B 19 3.374 18.496 34.286 1.00 9.96 C ATOM 3641 OH TYR B 19 2.725 19.608 33.802 1.00 11.01 O ATOM 3643 CE2 TYR B 19 3.987 18.565 35.519 1.00 10.79 C ATOM 3645 CD2 TYR B 19 4.660 17.462 36.020 1.00 10.02 C ATOM 3647 C TYR B 19 4.951 12.801 36.969 1.00 7.80 C ATOM 3648 O TYR B 19 4.984 11.860 36.180 1.00 8.04 O ATOM 3649 N ARG B 20 5.385 12.701 38.224 1.00 7.62 N ATOM 3651 CA ARG B 20 5.919 11.452 38.741 1.00 7.92 C ATOM 3653 CB ARG B 20 6.659 11.679 40.056 1.00 8.70 C ATOM 3656 CG ARG B 20 5.865 12.292 41.176 1.00 9.58 C ATOM 3659 CD ARG B 20 6.640 12.228 42.469 1.00 10.61 C ATOM 3662 NE ARG B 20 5.937 12.768 43.620 1.00 12.27 N ATOM 3664 CZ ARG B 20 6.343 13.830 44.332 1.00 14.55 C ATOM 3665 NH1 ARG B 20 7.433 14.528 44.011 1.00 15.43 N ATOM 3668 NH2 ARG B 20 5.641 14.205 45.395 1.00 15.98 N ATOM 3671 C ARG B 20 4.833 10.398 38.938 1.00 7.88 C ATOM 3672 O ARG B 20 5.142 9.210 39.062 1.00 8.74 O ATOM 3673 N ALA B 21 3.573 10.834 38.989 1.00 7.67 N ATOM 3675 CA ALA B 21 2.436 9.931 39.101 1.00 7.77 C ATOM 3677 CB ALA B 21 1.355 10.545 40.004 1.00 8.33 C ATOM 3681 C ALA B 21 1.860 9.554 37.740 1.00 7.49 C ATOM 3682 O ALA B 21 0.883 8.813 37.670 1.00 8.24 O ATOM 3683 N ILE B 22 2.451 10.077 36.668 1.00 7.07 N ATOM 3685 CA ILE B 22 2.180 9.629 35.315 1.00 7.15 C ATOM 3687 CB ILE B 22 2.239 10.805 34.320 1.00 7.19 C ATOM 3689 CG1 ILE B 22 1.204 11.861 34.727 1.00 7.74 C ATOM 3692 CD1 ILE B 22 1.150 13.060 33.823 1.00 7.78 C ATOM 3696 CG2 ILE B 22 2.012 10.301 32.895 1.00 7.55 C ATOM 3700 C ILE B 22 3.192 8.540 35.014 1.00 7.08 C ATOM 3701 O ILE B 22 4.376 8.686 35.297 1.00 8.15 O ATOM 3702 N VAL B 23 2.708 7.426 34.477 1.00 7.33 N ATOM 3704 CA VAL B 23 3.505 6.221 34.384 1.00 7.49 C ATOM 3706 CB VAL B 23 2.933 5.092 35.284 1.00 7.65 C ATOM 3708 CG1 VAL B 23 2.619 5.599 36.672 1.00 8.69 C ATOM 3712 CG2 VAL B 23 1.690 4.436 34.682 1.00 8.21 C ATOM 3716 C VAL B 23 3.625 5.760 32.939 1.00 6.99 C ATOM 3717 O VAL B 23 2.710 5.912 32.130 1.00 7.44 O ATOM 3718 N HIS B 24 4.788 5.194 32.623 1.00 7.09 N ATOM 3720 CA HIS B 24 5.005 4.494 31.375 1.00 7.24 C ATOM 3722 CB HIS B 24 6.484 4.596 30.984 1.00 7.56 C ATOM 3725 CG HIS B 24 6.810 3.808 29.779 1.00 8.11 C ATOM 3726 ND1 HIS B 24 7.112 2.467 29.831 1.00 9.52 N ATOM 3728 CE1 HIS B 24 7.263 2.022 28.599 1.00 10.58 C ATOM 3730 NE2 HIS B 24 7.090 3.026 27.757 1.00 11.37 N ATOM 3732 CD2 HIS B 24 6.804 4.156 28.474 1.00 10.43 C ATOM 3734 C HIS B 24 4.599 3.027 31.568 1.00 7.57 C ATOM 3735 O HIS B 24 4.949 2.409 32.577 1.00 8.17 O ATOM 3736 N ILE B 25 3.848 2.485 30.615 1.00 7.37 N ATOM 3738 CA ILE B 25 3.381 1.108 30.652 1.00 7.87 C ATOM 3740 CB ILE B 25 1.842 1.058 30.651 1.00 8.18 C ATOM 3742 CG1 ILE B 25 1.257 1.843 31.824 1.00 9.00 C ATOM 3745 CD1 ILE B 25 −0.242 2.093 31.705 1.00 8.99 C ATOM 3749 CG2 ILE B 25 1.356 −0.398 30.666 1.00 9.66 C ATOM 3753 C ILE B 25 3.899 0.364 29.441 1.00 8.15 C ATOM 3754 O ILE B 25 3.755 0.843 28.315 1.00 8.94 O ATOM 3755 N SER B 26 4.486 −0.806 29.669 1.00 8.77 N ATOM 3757 CA SER B 26 4.773 −1.727 28.581 1.00 9.89 C ATOM 3759 CB BSER B 26 6.238 −1.804 28.196 0.35 10.66 C ATOM 3760 CB ASER B 26 6.305 −1.864 28.514 0.65 11.47 C ATOM 3765 OG BSER B 26 6.986 −2.328 29.246 0.35 11.77 O ATOM 3766 OG ASER B 26 6.755 −2.916 27.701 0.65 12.82 O ATOM 3769 C SER B 26 4.177 −3.089 28.889 1.00 9.15 C ATOM 3770 O SER B 26 4.245 −3.579 30.017 1.00 9.90 O ATOM 3771 N SER B 27 3.579 −3.695 27.878 1.00 8.91 N ATOM 3773 CA SER B 27 3.049 −5.042 27.993 1.00 9.24 C ATOM 3775 CB SER B 27 1.609 −5.020 28.523 1.00 9.75 C ATOM 3778 OG SER B 27 0.701 −4.659 27.498 1.00 10.07 O ATOM 3780 C SER B 27 3.045 −5.686 26.626 1.00 9.09 C ATOM 3781 O SER B 27 3.418 −5.071 25.633 1.00 9.64 O ATOM 3782 N SER B 28 2.555 −6.913 26.573 1.00 9.24 N ATOM 3784 CA SER B 28 2.448 −7.620 25.319 1.00 9.63 C ATOM 3786 CB SER B 28 1.950 −9.034 25.569 1.00 10.05 C ATOM 3789 OG SER B 28 0.663 −9.022 26.149 1.00 11.00 O ATOM 3791 C SER B 28 1.551 −6.906 24.309 1.00 9.09 C ATOM 3792 O SER B 28 1.683 −7.141 23.109 1.00 10.26 O ATOM 3793 N ILE B 29 0.612 −6.081 24.765 1.00 9.01 N ATOM 3795 CA ILE B 29 −0.230 −5.322 23.829 1.00 9.45 C ATOM 3797 CB ILE B 29 −1.528 −4.860 24.527 1.00 9.84 C ATOM 3799 CG1 ILE B 29 −2.467 −6.054 24.687 1.00 10.68 C ATOM 3802 CD1 ILE B 29 −3.749 −5.729 25.407 1.00 11.23 C ATOM 3806 CG2 ILE B 29 −2.209 −3.738 23.755 1.00 10.93 C ATOM 3810 C ILE B 29 0.520 −4.165 23.182 1.00 9.75 C ATOM 3811 O ILE B 29 0.298 −3.856 22.009 1.00 10.61 O ATOM 3812 N GLY B 30 1.392 −3.519 23.936 1.00 9.50 N ATOM 3814 CA GLY B 30 2.104 −2.366 23.439 1.00 10.18 C ATOM 3817 C GLY B 30 2.498 −1.451 24.564 1.00 8.93 C ATOM 3818 O GLY B 30 2.432 −1.827 25.728 1.00 10.65 O ATOM 3819 N SER B 31 2.926 −0.258 24.195 1.00 9.21 N ATOM 3821 CA SER B 31 3.322 0.746 25.151 1.00 9.76 C ATOM 3823 CB BSER B 31 4.627 1.413 24.672 0.35 10.79 C ATOM 3824 CB ASER B 31 4.636 1.385 24.762 0.65 11.07 C ATOM 3829 OG BSER B 31 5.007 2.545 25.442 0.35 12.74 O ATOM 3830 OG ASER B 31 5.642 0.393 24.813 0.65 12.96 O ATOM 3833 C SER B 31 2.236 1.796 25.263 1.00 8.79 C ATOM 3834 O SER B 31 1.624 2.194 24.261 1.00 10.03 O ATOM 3835 N CYS B 32 2.006 2.249 26.481 1.00 8.21 N ATOM 3837 CA CYS B 32 0.981 3.237 26.755 1.00 8.25 C ATOM 3839 CB BCYS B 32 −0.398 2.638 26.853 0.35 9.91 C ATOM 3840 CB ACYS B 32 −0.338 2.497 27.106 0.65 8.79 C ATOM 3845 SG BCYS B 32 −0.604 1.615 28.261 0.35 14.50 S ATOM 3846 SG ACYS B 32 −1.274 1.895 25.659 0.65 7.95 S ATOM 3847 C CYS B 32 1.399 4.076 27.956 1.00 7.16 C ATOM 3848 O CYS B 32 2.526 3.975 28.467 1.00 8.13 O ATOM 3849 N THR B 33 0.491 4.947 28.359 1.00 6.54 N ATOM 3851 CA THR B 33 0.647 5.783 29.522 1.00 6.41 C ATOM 3853 CB THR B 33 0.515 7.251 29.080 1.00 6.34 C ATOM 3855 OG1 THR B 33 1.515 7.524 28.079 1.00 6.92 O ATOM 3857 CG2 THR B 33 0.761 8.237 30.220 1.00 6.68 C ATOM 3861 C THR B 33 −0.451 5.417 30.520 1.00 6.49 C ATOM 3862 O THR B 33 −1.496 4.893 30.137 1.00 6.80 O ATOM 3863 N GLY B 34 −0.228 5.715 31.793 1.00 6.76 N ATOM 3865 CA GLY B 34 −1.290 5.682 32.779 1.00 6.72 C ATOM 3868 C GLY B 34 −1.039 6.736 33.827 1.00 6.52 C ATOM 3869 O GLY B 34 −0.075 7.493 33.760 1.00 6.78 O ATOM 3870 N TRP B 35 −1.887 6.753 34.838 1.00 6.86 N ATOM 3872 CA TRP B 35 −1.766 7.724 35.904 1.00 7.26 C ATOM 3874 CB TRP B 35 −2.492 9.043 35.563 1.00 7.82 C ATOM 3877 CG TRP B 35 −3.831 8.901 34.906 1.00 8.11 C ATOM 3878 CD1 TRP B 35 −4.066 8.555 33.608 1.00 8.12 C ATOM 3880 NE1 TRP B 35 −5.414 8.580 33.339 1.00 8.93 N ATOM 3882 CE2 TRP B 35 −6.079 8.965 34.473 1.00 8.81 C ATOM 3883 CD2 TRP B 35 −5.111 9.181 35.475 1.00 7.96 C ATOM 3884 CE3 TRP B 35 −5.542 9.590 36.735 1.00 8.75 C ATOM 3886 CZ3 TRP B 35 −6.887 9.760 36.966 1.00 9.89 C ATOM 3888 CH2 TRP B 35 −7.814 9.526 35.963 1.00 10.09 C ATOM 3890 CZ2 TRP B 35 −7.432 9.140 34.705 1.00 10.05 C ATOM 3892 C TRP B 35 −2.265 7.119 37.203 1.00 7.17 C ATOM 3893 O TRP B 35 −3.305 6.444 37.247 1.00 7.48 O ATOM 3894 N MET B 36 −1.514 7.324 38.276 1.00 7.22 N ATOM 3896 CA MET B 36 −1.884 6.750 39.562 1.00 7.60 C ATOM 3898 CB MET B 36 −0.790 6.983 40.601 1.00 8.12 C ATOM 3901 CG MET B 36 0.593 6.429 40.265 1.00 8.68 C ATOM 3904 SD MET B 36 0.683 4.684 39.895 1.00 9.14 S ATOM 3905 CE MET B 36 0.098 4.015 41.440 1.00 9.93 C ATOM 3909 C MET B 36 −3.173 7.378 40.084 1.00 7.70 C ATOM 3910 O MET B 36 −3.339 8.603 40.029 1.00 8.47 O ATOM 3911 N ILE B 37 −4.055 6.534 40.632 1.00 7.60 N ATOM 3913 CA ILE B 37 −5.248 6.992 41.337 1.00 8.62 C ATOM 3915 CB ILE B 37 −6.553 6.614 40.591 1.00 8.72 C ATOM 3917 CG1 ILE B 37 −6.723 5.099 40.438 1.00 9.33 C ATOM 3920 CD1 ILE B 37 −8.120 4.724 39.928 1.00 9.73 C ATOM 3924 CG2 ILE B 37 −6.607 7.330 39.261 1.00 9.21 C ATOM 3928 C ILE B 37 −5.294 6.519 42.789 1.00 8.85 C ATOM 3929 O ILE B 37 −6.214 6.872 43.524 1.00 10.47 O ATOM 3930 N GLY B 38 −4.311 5.739 43.210 1.00 9.34 N ATOM 3932 CA GLY B 38 −4.205 5.289 44.585 1.00 9.66 C ATOM 3935 C GLY B 38 −2.837 4.675 44.794 1.00 9.97 C ATOM 3936 O GLY B 38 −1.986 4.723 43.900 1.00 10.35 O ATOM 3937 N PRO B 39 −2.597 4.131 45.975 1.00 9.86 N ATOM 3938 CA PRO B 39 −1.304 3.498 46.274 1.00 10.14 C ATOM 3940 CB PRO B 39 −1.552 2.839 47.634 1.00 10.75 C ATOM 3943 CG PRO B 39 −2.545 3.766 48.271 1.00 11.80 C ATOM 3946 CD PRO B 39 −3.486 4.139 47.149 1.00 10.25 C ATOM 3949 C PRO B 39 −0.830 2.487 45.238 1.00 9.69 C ATOM 3950 O PRO B 39 0.366 2.411 44.978 1.00 10.04 O ATOM 3951 N LYS B 40 −1.734 1.687 44.702 1.00 9.60 N ATOM 3953 CA LYS B 40 −1.328 0.634 43.791 1.00 9.71 C ATOM 3955 CB LYS B 40 −1.113 −0.678 44.529 1.00 11.09 C ATOM 3958 CG LYS B 40 −2.335 −1.186 45.229 1.00 11.94 C ATOM 3961 CD LYS B 40 −2.132 −2.615 45.726 1.00 13.45 C ATOM 3964 CE LYS B 40 −0.996 −2.749 46.704 1.00 14.20 C ATOM 3967 NZ LYS B 40 −0.976 −4.121 47.344 1.00 15.10 N ATOM 3971 C LYS B 40 −2.284 0.467 42.617 1.00 8.70 C ATOM 3972 O LYS B 40 −2.366 −0.617 42.060 1.00 9.87 O ATOM 3973 N THR B 41 −2.985 1.532 42.227 1.00 8.11 N ATOM 3975 CA THR B 41 −3.939 1.455 41.125 1.00 8.14 C ATOM 3977 CB THR B 41 −5.375 1.586 41.663 1.00 8.25 C ATOM 3979 OG1 THR B 41 −5.572 0.652 42.741 1.00 9.37 O ATOM 3981 CG2 THR B 41 −6.399 1.262 40.576 1.00 9.16 C ATOM 3985 C THR B 41 −3.641 2.556 40.130 1.00 7.63 C ATOM 3986 O THR B 41 −3.476 3.711 40.515 1.00 8.27 O ATOM 3987 N VAL B 42 −3.590 2.160 38.861 1.00 7.48 N ATOM 3989 CA VAL B 42 −3.271 3.007 37.732 1.00 7.56 C ATOM 3991 CB VAL B 42 −2.122 2.378 36.911 1.00 7.80 C ATOM 3993 CG1 VAL B 42 −1.745 3.260 35.729 1.00 8.94 C ATOM 3997 CG2 VAL B 42 −0.914 2.085 37.763 1.00 9.62 C ATOM 4001 C VAL B 42 −4.491 3.072 36.818 1.00 7.34 C ATOM 4002 O VAL B 42 −5.024 2.044 36.433 1.00 9.14 O ATOM 4003 N ALA B 43 −4.918 4.274 36.432 1.00 7.37 N ATOM 4005 CA ALA B 43 −5.911 4.442 35.377 1.00 7.20 C ATOM 4007 CB ALA B 43 −6.711 5.713 35.603 1.00 7.51 C ATOM 4011 C ALA B 43 −5.214 4.503 34.017 1.00 7.00 C ATOM 4012 O ALA B 43 −4.129 5.081 33.886 1.00 7.26 O ATOM 4013 N THR B 44 −5.836 3.904 33.019 1.00 6.97 N ATOM 4015 CA THR B 44 −5.286 3.897 31.670 1.00 7.04 C ATOM 4017 CB THR B 44 −4.160 2.834 31.570 1.00 7.41 C ATOM 4019 OG1 THR B 44 −3.485 2.938 30.303 1.00 7.54 O ATOM 4021 CG2 THR B 44 −4.692 1.413 31.698 1.00 7.72 C ATOM 4025 C THR B 44 −6.413 3.683 30.656 1.00 6.99 C ATOM 4026 O THR B 44 −7.596 3.731 30.998 1.00 7.52 O ATOM 4027 N ALA B 45 −6.048 3.485 29.395 1.00 7.00 N ATOM 4029 CA ALA B 45 −7.003 3.149 28.349 1.00 7.12 C ATOM 4031 CB ALA B 45 −6.479 3.579 26.979 1.00 7.53 C ATOM 4035 C ALA B 45 −7.281 1.644 28.351 1.00 7.28 C ATOM 4036 O ALA B 45 −6.370 0.833 28.543 1.00 8.36 O ATOM 4037 N GLY B 46 −8.529 1.256 28.120 1.00 7.41 N ATOM 4039 CA GLY B 46 −8.874 −0.156 28.014 1.00 7.78 C ATOM 4042 C GLY B 46 −8.106 −0.884 26.933 1.00 7.87 C ATOM 4043 O GLY B 46 −7.669 −2.017 27.135 1.00 8.48 O ATOM 4044 N HIS B 47 −7.940 −0.234 25.783 1.00 7.88 N ATOM 4046 CA HIS B 47 −7.288 −0.893 24.672 1.00 8.40 C ATOM 4048 CB HIS B 47 −7.524 −0.133 23.362 1.00 8.56 C ATOM 4051 CG HIS B 47 −6.718 1.122 23.182 1.00 7.89 C ATOM 4052 ND1 HIS B 47 −7.280 2.381 23.233 1.00 8.37 N ATOM 4054 CE1 HIS B 47 −6.356 3.284 22.954 1.00 8.17 C ATOM 4056 NE2 HIS B 47 −5.209 2.668 22.753 1.00 8.05 N ATOM 4058 CD2 HIS B 47 −5.409 1.313 22.884 1.00 7.79 C ATOM 4060 C HIS B 47 −5.808 −1.162 24.909 1.00 8.34 C ATOM 4061 O HIS B 47 −5.198 −1.909 24.160 1.00 9.86 O ATOM 4062 N CYS B 48 −5.235 −0.537 25.933 1.00 7.91 N ATOM 4064 CA CYS B 48 −3.850 −0.803 26.311 1.00 8.43 C ATOM 4066 CB CYS B 48 −3.317 0.340 27.164 1.00 9.43 C ATOM 4069 SG CYS B 48 −3.197 1.908 26.286 1.00 11.14 S ATOM 4070 C CYS B 48 −3.671 −2.102 27.099 1.00 8.41 C ATOM 4071 O CYS B 48 −2.553 −2.599 27.197 1.00 9.30 O ATOM 4072 N ILE B 49 −4.758 −2.622 27.679 1.00 8.25 N ATOM 4074 CA ILE B 49 −4.680 −3.771 28.589 1.00 8.11 C ATOM 4076 CB ILE B 49 −4.931 −3.327 30.049 1.00 8.38 C ATOM 4078 CG1 ILE B 49 −6.349 −2.791 30.254 1.00 8.89 C ATOM 4081 CD1 ILE B 49 −6.631 −2.365 31.696 1.00 9.33 C ATOM 4085 CG2 ILE B 49 −3.871 −2.314 30.454 1.00 9.04 C ATOM 4089 C ILE B 49 −5.574 −4.945 28.224 1.00 8.36 C ATOM 4090 O ILE B 49 −5.385 −6.015 28.774 1.00 8.42 O ATOM 4091 N TYR B 50 −6.527 −4.765 27.313 1.00 8.78 N ATOM 4093 CA TYR B 50 −7.397 −5.847 26.876 1.00 9.04 C ATOM 4095 CB TYR B 50 −8.752 −5.812 27.602 1.00 9.41 C ATOM 4098 CG TYR B 50 −9.689 −6.905 27.142 1.00 10.04 C ATOM 4099 CD1 TYR B 50 −10.686 −6.650 26.211 1.00 10.86 C ATOM 4101 CE1 TYR B 50 −11.534 −7.668 25.770 1.00 11.77 C ATOM 4103 CZ TYR B 50 −11.372 −8.951 26.279 1.00 11.98 C ATOM 4104 OH TYR B 50 −12.188 −9.993 25.878 1.00 14.06 O ATOM 4106 CE2 TYR B 50 −10.394 −9.208 27.210 1.00 11.89 C ATOM 4108 CD2 TYR B 50 −9.549 −8.200 27.615 1.00 10.91 C ATOM 4110 C TYR B 50 −7.585 −5.731 25.363 1.00 9.64 C ATOM 4111 O TYR B 50 −8.007 −4.678 24.858 1.00 10.03 O ATOM 4112 N ASP B 51 −7.221 −6.802 24.663 1.00 10.47 N ATOM 4114 CA ASP B 51 −7.291 −6.906 23.220 1.00 12.23 C ATOM 4116 CB BASP B 51 −6.107 −7.742 22.729 0.35 12.66 C ATOM 4117 CB AASP B 51 −6.122 −7.695 22.640 0.65 13.16 C ATOM 4122 CG BASP B 51 −6.080 −7.888 21.234 0.35 13.82 C ATOM 4123 CG AASP B 51 −6.149 −7.713 21.131 0.65 15.14 C ATOM 4124 OD1 BASP B 51 −6.122 −9.033 20.747 0.35 14.80 O ATOM 4125 OD1 AASP B 51 −5.098 −7.505 20.497 0.65 16.90 O ATOM 4126 OD2 BASP B 51 −6.018 −6.909 20.468 0.35 15.44 O ATOM 4127 OD2 AASP B 51 −7.200 −7.900 20.492 0.65 16.43 O ATOM 4128 C ASP B 51 −8.601 −7.577 22.843 1.00 11.68 C ATOM 4129 O ASP B 51 −8.809 −8.770 23.089 1.00 12.14 O ATOM 4130 N THR B 52 −9.484 −6.811 22.224 1.00 12.82 N ATOM 4132 CA THR B 52 −10.821 −7.311 21.944 1.00 14.29 C ATOM 4134 CB THR B 52 −11.794 −6.158 21.621 1.00 15.31 C ATOM 4136 OG1 THR B 52 −11.342 −5.436 20.473 1.00 17.85 O ATOM 4138 CG2 THR B 52 −11.813 −5.133 22.748 1.00 15.84 C ATOM 4142 C THR B 52 −10.849 −8.374 20.842 1.00 15.07 C ATOM 4143 O THR B 52 −11.736 −9.221 20.836 1.00 16.91 O ATOM 4144 N SER B 53 −9.900 −8.338 19.911 1.00 15.21 N ATOM 4146 CA SER B 53 −9.869 −9.326 18.824 1.00 15.87 C ATOM 4148 CB BSER B 53 −8.908 −8.886 17.708 0.35 16.21 C ATOM 4149 CB ASER B 53 −8.859 −8.903 17.756 0.65 16.72 C ATOM 4154 OG BSER B 53 −7.569 −8.772 18.157 0.35 17.00 O ATOM 4155 OG ASER B 53 −8.752 −9.892 16.748 0.65 18.99 O ATOM 4158 C SER B 53 −9.530 −10.736 19.309 1.00 15.03 C ATOM 4159 O SER B 53 −10.178 −11.722 18.919 1.00 14.93 O ATOM 4160 N SER B 54 −8.511 −10.836 20.153 1.00 14.11 N ATOM 4162 CA SER B 54 −8.082 −12.117 20.691 1.00 13.76 C ATOM 4164 CB SER B 54 −6.585 −12.082 20.984 1.00 14.63 C ATOM 4167 OG SER B 54 −6.302 −11.212 22.069 1.00 15.48 O ATOM 4169 C SER B 54 −8.830 −12.497 21.955 1.00 12.67 C ATOM 4170 O SER B 54 −8.716 −13.624 22.416 1.00 13.34 O ATOM 4171 N GLY B 55 −9.564 −11.539 22.518 1.00 12.60 N ATOM 4173 CA GLY B 55 −10.337 −11.766 23.724 1.00 12.43 C ATOM 4176 C GLY B 55 −9.474 −11.987 24.936 1.00 11.80 C ATOM 4177 O GLY B 55 −9.834 −12.737 25.833 1.00 12.09 O ATOM 4178 N SER B 56 −8.333 −11.313 24.993 1.00 12.30 N ATOM 4180 CA SER B 56 −7.404 −11.563 26.071 1.00 12.22 C ATOM 4182 CB SER B 56 −6.277 −12.470 25.600 1.00 13.33 C ATOM 4185 OG SER B 56 −5.511 −11.840 24.607 1.00 17.47 O ATOM 4187 C SER B 56 −6.813 −10.288 26.619 1.00 10.81 C ATOM 4188 O SER B 56 −6.567 −9.310 25.907 1.00 10.50 O ATOM 4189 N PHE B 57 −6.573 −10.325 27.916 1.00 9.99 N ATOM 4191 CA PHE B 57 −5.790 −9.301 28.562 1.00 9.43 C ATOM 4193 CB PHE B 57 −5.887 −9.455 30.080 1.00 10.07 C ATOM 4196 CG PHE B 57 −7.232 −9.069 30.620 1.00 10.41 C ATOM 4197 CD1 PHE B 57 −7.527 −7.744 30.869 1.00 10.08 C ATOM 4199 CE1 PHE B 57 −8.774 −7.363 31.333 1.00 11.19 C ATOM 4201 CZ PHE B 57 −9.751 −8.313 31.532 1.00 12.88 C ATOM 4203 CE2 PHE B 57 −9.476 −9.645 31.264 1.00 13.00 C ATOM 4205 CD2 PHE B 57 −8.230 −10.020 30.810 1.00 12.20 C ATOM 4207 C PHE B 57 −4.347 −9.410 28.102 1.00 9.19 C ATOM 4208 O PHE B 57 −3.877 −10.475 27.678 1.00 10.24 O ATOM 4209 N ALA B 58 −3.643 −8.288 28.189 1.00 9.20 N ATOM 4211 CA ALA B 58 −2.202 −8.292 28.075 1.00 9.09 C ATOM 4213 CB ALA B 58 −1.664 −6.887 28.322 1.00 9.63 C ATOM 4217 C ALA B 58 −1.601 −9.247 29.090 1.00 9.25 C ATOM 4218 O ALA B 58 −2.213 −9.573 30.105 1.00 9.38 O ATOM 4219 N GLY B 59 −0.371 −9.666 28.838 1.00 9.59 N ATOM 4221 CA GLY B 59 0.444 −10.276 29.857 1.00 9.95 C ATOM 4224 C GLY B 59 0.793 −9.242 30.908 1.00 9.76 C ATOM 4225 O GLY B 59 0.308 −8.099 30.891 1.00 10.29 O ATOM 4226 N THR B 60 1.637 −9.646 31.834 1.00 10.02 N ATOM 4228 CA THR B 60 2.060 −8.759 32.898 1.00 10.25 C ATOM 4230 CB THR B 60 3.107 −9.463 33.740 1.00 11.48 C ATOM 4232 OG1 THR B 60 2.519 −10.662 34.262 1.00 13.35 O ATOM 4234 CG2 THR B 60 3.526 −8.622 34.941 1.00 12.09 C ATOM 4238 C THR B 60 2.629 −7.471 32.338 1.00 9.81 C ATOM 4239 O THR B 60 3.465 −7.498 31.441 1.00 10.64 O ATOM 4240 N ALA B 61 2.176 −6.351 32.884 1.00 9.32 N ATOM 4242 CA ALA B 61 2.677 −5.044 32.503 1.00 9.32 C ATOM 4244 CB ALA B 61 1.568 −3.981 32.587 1.00 9.62 C ATOM 4248 C ALA B 61 3.837 −4.632 33.385 1.00 8.92 C ATOM 4249 O ALA B 61 3.876 −4.954 34.567 1.00 10.09 O ATOM 4250 N THR B 62 4.756 −3.882 32.793 1.00 9.06 N ATOM 4252 CA THR B 62 5.844 −3.224 33.497 1.00 9.56 C ATOM 4254 CB THR B 62 7.159 −3.456 32.762 1.00 10.57 C ATOM 4256 OG1 THR B 62 7.423 −4.870 32.721 1.00 11.83 O ATOM 4258 CG2 THR B 62 8.326 −2.808 33.497 1.00 12.14 C ATOM 4262 C THR B 62 5.495 −1.745 33.556 1.00 8.59 C ATOM 4263 O THR B 62 5.334 −1.089 32.521 1.00 9.17 O ATOM 4264 N VAL B 63 5.359 −1.225 34.771 1.00 8.26 N ATOM 4266 CA VAL B 63 4.826 0.118 35.013 1.00 8.04 C ATOM 4268 CB VAL B 63 3.546 0.039 35.861 1.00 8.65 C ATOM 4270 CG1 VAL B 63 3.023 1.431 36.176 1.00 9.71 C ATOM 4274 CG2 VAL B 63 2.478 −0.794 35.150 1.00 9.51 C ATOM 4278 C VAL B 63 5.891 0.959 35.693 1.00 7.95 C ATOM 4279 O VAL B 63 6.369 0.597 36.771 1.00 8.82 O ATOM 4280 N SER B 64 6.254 2.083 35.085 1.00 7.68 N ATOM 4282 CA SER B 64 7.393 2.863 35.515 1.00 8.03 C ATOM 4284 CB SER B 64 8.499 2.805 34.462 1.00 8.70 C ATOM 4287 OG SER B 64 8.898 1.469 34.228 1.00 9.66 O ATOM 4289 C SER B 64 6.965 4.306 35.757 1.00 7.95 C ATOM 4290 O SER B 64 6.893 5.116 34.823 1.00 7.83 O ATOM 4291 N PRO B 65 6.648 4.658 37.004 1.00 8.11 N ATOM 4292 CA PRO B 65 6.226 6.028 37.301 1.00 8.10 C ATOM 4294 CB PRO B 65 5.859 5.970 38.795 1.00 8.49 C ATOM 4297 CG PRO B 65 5.584 4.520 39.054 1.00 8.49 C ATOM 4300 CD PRO B 65 6.600 3.807 38.204 1.00 8.68 C ATOM 4303 C PRO B 65 7.344 7.027 37.057 1.00 8.00 C ATOM 4304 O PRO B 65 8.483 6.807 37.481 1.00 8.46 O ATOM 4305 N GLY B 66 7.038 8.127 36.383 1.00 7.75 N ATOM 4307 CA GLY B 66 8.034 9.166 36.186 1.00 8.40 C ATOM 4310 C GLY B 66 9.266 8.699 35.428 1.00 8.24 C ATOM 4311 O GLY B 66 10.346 9.265 35.586 1.00 8.86 O ATOM 4312 N ARG B 67 9.123 7.685 34.585 1.00 8.08 N ATOM 4314 CA ARG B 67 10.223 7.252 33.745 1.00 8.11 C ATOM 4316 CB ARG B 67 9.753 6.160 32.802 1.00 8.27 C ATOM 4319 CG ARG B 67 10.864 5.568 31.971 1.00 8.88 C ATOM 4322 CD ARG B 67 10.435 4.444 31.086 1.00 8.89 C ATOM 4325 NE ARG B 67 11.498 4.135 30.142 1.00 9.16 N ATOM 4327 CZ ARG B 67 11.404 3.282 29.149 1.00 10.30 C ATOM 4328 NH1 ARG B 67 12.410 3.169 28.296 1.00 11.25 N ATOM 4331 NH2 ARG B 67 10.320 2.541 29.004 1.00 12.36 N ATOM 4334 C ARG B 67 10.750 8.429 32.946 1.00 8.11 C ATOM 4335 O ARG B 67 9.983 9.254 32.462 1.00 8.22 O ATOM 4336 N ASN B 68 12.070 8.472 32.783 1.00 8.17 N ATOM 4338 CA ASN B 68 12.720 9.484 31.970 1.00 8.77 C ATOM 4340 CB ASN B 68 13.312 10.573 32.848 1.00 9.40 C ATOM 4343 CG ASN B 68 13.931 11.660 32.023 1.00 10.39 C ATOM 4344 OD1 ASN B 68 13.349 12.050 31.010 1.00 11.79 O ATOM 4345 ND2 ASN B 68 15.136 12.110 32.385 1.00 12.51 N ATOM 4348 C ASN B 68 13.812 8.863 31.104 1.00 9.03 C ATOM 4349 O ASN B 68 14.994 8.879 31.455 1.00 9.74 O ATOM 4350 N GLY B 69 13.405 8.293 29.977 1.00 9.60 N ATOM 4352 CA GLY B 69 14.329 7.682 29.037 1.00 9.82 C ATOM 4355 C GLY B 69 14.763 6.335 29.549 1.00 9.55 C ATOM 4356 O GLY B 69 13.946 5.419 29.628 1.00 10.48 O ATOM 4357 N THR B 70 16.040 6.194 29.885 1.00 9.50 N ATOM 4359 CA THR B 70 16.516 4.977 30.529 1.00 10.01 C ATOM 4361 CB THR B 70 17.775 4.427 29.839 1.00 10.64 C ATOM 4363 OG1 THR B 70 18.745 5.471 29.679 1.00 11.68 O ATOM 4365 CG2 THR B 70 17.437 3.934 28.436 1.00 11.69 C ATOM 4369 C THR B 70 16.747 5.185 32.024 1.00 10.48 C ATOM 4370 O THR B 70 17.362 4.357 32.689 1.00 11.63 O ATOM 4371 N SER B 71 16.214 6.274 32.558 1.00 10.58 N ATOM 4373 CA SER B 71 16.175 6.510 33.992 1.00 10.32 C ATOM 4375 CB SER B 71 16.437 7.969 34.309 1.00 10.45 C ATOM 4378 OG SER B 71 17.669 8.393 33.780 1.00 11.02 O ATOM 4380 C SER B 71 14.821 6.139 34.562 1.00 9.95 C ATOM 4381 O SER B 71 13.775 6.496 34.006 1.00 10.07 O ATOM 4382 N TYR B 72 14.853 5.470 35.710 1.00 9.77 N ATOM 4384 CA TYR B 72 13.665 4.953 36.370 1.00 9.83 C ATOM 4386 CB TYR B 72 13.637 3.420 36.298 1.00 10.11 C ATOM 4389 CG TYR B 72 13.491 2.884 34.890 1.00 10.38 C ATOM 4390 CD1 TYR B 72 12.261 2.467 34.422 1.00 10.73 C ATOM 4392 CE1 TYR B 72 12.112 1.963 33.142 1.00 11.24 C ATOM 4394 CZ TYR B 72 13.200 1.895 32.301 1.00 11.23 C ATOM 4395 OH TYR B 72 13.014 1.381 31.041 1.00 12.73 O ATOM 4397 CE2 TYR B 72 14.442 2.316 32.741 1.00 11.74 C ATOM 4399 CD2 TYR B 72 14.581 2.804 34.018 1.00 11.15 C ATOM 4401 C TYR B 72 13.739 5.443 37.815 1.00 10.00 C ATOM 4402 O TYR B 72 14.125 4.683 38.712 1.00 10.50 O ATOM 4403 N PRO B 73 13.426 6.715 38.070 1.00 10.19 N ATOM 4404 CA PRO B 73 13.605 7.254 39.425 1.00 10.57 C ATOM 4406 CB PRO B 73 13.195 8.719 39.285 1.00 10.64 C ATOM 4409 CG PRO B 73 12.351 8.766 38.059 1.00 10.69 C ATOM 4412 CD PRO B 73 12.927 7.742 37.134 1.00 10.07 C ATOM 4415 C PRO B 73 12.778 6.561 40.497 1.00 10.59 C ATOM 4416 O PRO B 73 13.139 6.627 41.664 1.00 12.17 O ATOM 4417 N TYR B 74 11.692 5.916 40.097 1.00 10.13 N ATOM 4419 CA TYR B 74 10.834 5.165 41.004 1.00 10.86 C ATOM 4421 CB TYR B 74 9.425 5.767 41.038 1.00 10.82 C ATOM 4424 CG TYR B 74 9.500 7.222 41.399 1.00 10.36 C ATOM 4425 CD1 TYR B 74 9.391 8.194 40.416 1.00 10.82 C ATOM 4427 CE1 TYR B 74 9.519 9.518 40.701 1.00 11.59 C ATOM 4429 CZ TYR B 74 9.748 9.915 41.996 1.00 11.91 C ATOM 4430 OH TYR B 74 9.863 11.261 42.253 1.00 14.01 O ATOM 4432 CE2 TYR B 74 9.864 8.972 43.005 1.00 12.35 C ATOM 4434 CD2 TYR B 74 9.752 7.632 42.700 1.00 11.52 C ATOM 4436 C TYR B 74 10.788 3.696 40.635 1.00 11.39 C ATOM 4437 O TYR B 74 9.849 2.993 41.013 1.00 12.84 O ATOM 4438 N GLY B 75 11.820 3.222 39.939 1.00 10.85 N ATOM 4440 CA GLY B 75 11.872 1.851 39.479 1.00 10.79 C ATOM 4443 C GLY B 75 10.764 1.505 38.505 1.00 10.56 C ATOM 4444 O GLY B 75 10.129 2.370 37.891 1.00 10.85 O ATOM 4445 N SER B 76 10.563 0.202 38.377 1.00 10.90 N ATOM 4447 CA SER B 76 9.489 −0.367 37.607 1.00 11.52 C ATOM 4449 CB BSER B 76 10.053 −1.085 36.386 0.35 11.19 C ATOM 4450 CB ASER B 76 9.998 −0.975 36.309 0.65 13.32 C ATOM 4455 OG BSER B 76 10.704 −0.188 35.508 0.35 7.99 O ATOM 4456 OG ASER B 76 10.880 −2.042 36.529 0.65 17.36 O ATOM 4459 C SER B 76 8.802 −1.393 38.474 1.00 11.22 C ATOM 4460 O SER B 76 9.444 −2.102 39.264 1.00 12.58 O ATOM 4461 N VAL B 77 7.489 −1.472 38.325 1.00 10.56 N ATOM 4463 CA VAL B 77 6.668 −2.352 39.116 1.00 10.65 C ATOM 4465 CB BVAL B 77 5.793 −1.531 40.080 0.35 10.56 C ATOM 4466 CB AVAL B 77 5.843 −1.555 40.151 0.65 11.99 C ATOM 4469 CG1 BVAL B 77 4.837 −2.397 40.810 0.35 8.39 C ATOM 4470 CG1 AVAL B 77 6.704 −0.441 40.775 0.65 12.64 C ATOM 4477 CG2 BVAL B 77 6.661 −0.843 41.119 0.35 11.33 C ATOM 4478 CG2 AVAL B 77 4.627 −0.943 39.578 0.65 12.49 C ATOM 4485 C VAL B 77 5.801 −3.183 38.174 1.00 9.91 C ATOM 4486 O VAL B 77 5.303 −2.699 37.163 1.00 12.13 O ATOM 4487 N LYS B 78 5.596 −4.440 38.500 1.00 11.93 N ATOM 4489 CA LYS B 78 4.790 −5.315 37.664 1.00 12.70 C ATOM 4491 CB LYS B 78 5.236 −6.767 37.809 1.00 13.36 C ATOM 4494 CG LYS B 78 6.666 −7.034 37.399 1.00 15.87 C ATOM 4497 CD LYS B 78 6.906 −6.736 35.938 1.00 17.52 C ATOM 4500 CE LYS B 78 8.294 −7.176 35.540 1.00 19.94 C ATOM 4503 NZ LYS B 78 8.671 −6.739 34.188 1.00 22.53 N ATOM 4507 C LYS B 78 3.338 −5.215 38.065 1.00 12.78 C ATOM 4508 O LYS B 78 3.035 −5.045 39.243 1.00 14.43 O ATOM 4509 N SER B 79 2.436 −5.360 37.098 1.00 12.03 N ATOM 4511 CA SER B 79 1.017 −5.509 37.378 1.00 11.74 C ATOM 4513 CB SER B 79 0.196 −5.437 36.090 1.00 11.51 C ATOM 4516 OG SER B 79 0.508 −6.477 35.178 1.00 10.77 O ATOM 4518 C SER B 79 0.722 −6.833 38.044 1.00 11.16 C ATOM 4519 O SER B 79 1.441 −7.826 37.856 1.00 12.07 O ATOM 4520 N THR B 80 −0.360 −6.849 38.804 1.00 10.75 N ATOM 4522 CA THR B 80 −0.923 −8.093 39.298 1.00 11.21 C ATOM 4524 CB THR B 80 −1.164 −8.015 40.807 1.00 11.49 C ATOM 4526 OG1 THR B 80 −1.989 −6.887 41.124 1.00 12.77 O ATOM 4528 CG2 THR B 80 0.154 −7.823 41.547 1.00 11.88 C ATOM 4532 C THR B 80 −2.196 −8.485 38.578 1.00 11.33 C ATOM 4533 O THR B 80 −2.490 −9.682 38.478 1.00 12.80 O ATOM 4534 N ARG B 81 −2.959 −7.489 38.114 1.00 11.02 N ATOM 4536 CA ARG B 81 −4.210 −7.752 37.427 1.00 10.96 C ATOM 4538 CB ARG B 81 −5.240 −8.338 38.374 1.00 11.73 C ATOM 4541 CG ARG B 81 −5.626 −7.375 39.459 1.00 11.26 C ATOM 4544 CD ARG B 81 −6.558 −7.993 40.419 1.00 13.29 C ATOM 4547 NE ARG B 81 −6.874 −7.102 41.525 1.00 14.74 N ATOM 4549 CZ ARG B 81 −7.891 −7.291 42.357 1.00 13.37 C ATOM 4550 NH1 ARG B 81 −8.139 −6.424 43.336 1.00 11.04 N ATOM 4553 NH2 ARG B 81 −8.704 −8.320 42.185 1.00 16.83 N ATOM 4556 C ARG B 81 −4.748 −6.458 36.824 1.00 9.86 C ATOM 4557 O ARG B 81 −4.234 −5.348 37.074 1.00 10.32 O ATOM 4558 N TYR B 82 −5.781 −6.619 36.013 1.00 9.05 N ATOM 4560 CA TYR B 82 −6.392 −5.564 35.243 1.00 8.45 C ATOM 4562 CB TYR B 82 −6.236 −5.882 33.761 1.00 8.46 C ATOM 4565 CG TYR B 82 −4.815 −5.913 33.273 1.00 8.62 C ATOM 4566 CD1 TYR B 82 −4.012 −4.791 33.367 1.00 9.06 C ATOM 4568 CE1 TYR B 82 −2.711 −4.804 32.888 1.00 9.18 C ATOM 4570 CZ TYR B 82 −2.202 −5.950 32.310 1.00 8.79 C ATOM 4571 OH TYR B 82 −0.907 −5.894 31.850 1.00 9.78 O ATOM 4573 CE2 TYR B 82 −2.990 −7.081 32.209 1.00 9.01 C ATOM 4575 CD2 TYR B 82 −4.284 −7.053 32.688 1.00 9.11 C ATOM 4577 C TYR B 82 −7.886 −5.476 35.560 1.00 8.75 C ATOM 4578 O TYR B 82 −8.513 −6.470 35.949 1.00 9.58 O ATOM 4579 N PHE B 83 −8.447 −4.290 35.362 1.00 8.61 N ATOM 4581 CA PHE B 83 −9.874 −4.032 35.444 1.00 8.66 C ATOM 4583 CB PHE B 83 −10.228 −3.092 36.585 1.00 9.00 C ATOM 4586 CG PHE B 83 −9.748 −3.516 37.936 1.00 9.24 C ATOM 4587 CD1 PHE B 83 −8.475 −3.177 38.366 1.00 10.26 C ATOM 4589 CE1 PHE B 83 −8.059 −3.502 39.639 1.00 11.44 C ATOM 4591 CZ PHE B 83 −8.911 −4.173 40.501 1.00 12.62 C ATOM 4593 CE2 PHE B 83 −10.177 −4.495 40.104 1.00 12.07 C ATOM 4595 CD2 PHE B 83 −10.604 −4.166 38.823 1.00 10.68 C ATOM 4597 C PHE B 83 −10.298 −3.339 34.160 1.00 8.76 C ATOM 4598 O PHE B 83 −9.630 −2.409 33.699 1.00 8.65 O ATOM 4599 N ILE B 84 −11.421 −3.768 33.598 1.00 8.84 N ATOM 4601 CA ILE B 84 −12.048 −3.068 32.478 1.00 9.12 C ATOM 4603 CB ILE B 84 −11.734 −3.740 31.118 1.00 9.53 C ATOM 4605 CG1 ILE B 84 −12.103 −5.225 31.124 1.00 10.28 C ATOM 4608 CD1 ILE B 84 −11.973 −5.909 29.791 1.00 12.10 C ATOM 4612 CG2 ILE B 84 −10.281 −3.522 30.746 1.00 10.12 C ATOM 4616 C ILE B 84 −13.552 −3.018 32.691 1.00 8.87 C ATOM 4617 O ILE B 84 −14.134 −3.904 33.327 1.00 9.55 O ATOM 4618 N PRO B 85 −14.198 −2.004 32.131 1.00 9.08 N ATOM 4619 CA PRO B 85 −15.660 −1.982 32.154 1.00 9.52 C ATOM 4621 CB PRO B 85 −15.984 −0.561 31.686 1.00 10.01 C ATOM 4624 CG PRO B 85 −14.849 −0.235 30.745 1.00 9.80 C ATOM 4627 CD PRO B 85 −13.642 −0.866 31.371 1.00 9.00 C ATOM 4630 C PRO B 85 −16.212 −3.010 31.176 1.00 10.10 C ATOM 4631 O PRO B 85 −15.561 −3.364 30.210 1.00 10.32 O ATOM 4632 N SER B 86 −17.437 −3.459 31.407 1.00 11.25 N ATOM 4634 CA SER B 86 −18.073 −4.415 30.502 1.00 12.53 C ATOM 4636 CB BSER B 86 −19.506 −4.715 30.963 0.35 13.18 C ATOM 4637 CB ASER B 86 −19.476 −4.789 30.986 0.65 13.91 C ATOM 4642 OG BSER B 86 −19.544 −5.098 32.327 0.35 14.94 O ATOM 4643 OG ASER B 86 −20.279 −3.644 31.135 0.65 17.06 O ATOM 4646 C SER B 86 −18.116 −3.886 29.071 1.00 12.33 C ATOM 4647 O SER B 86 −17.957 −4.654 28.127 1.00 13.62 O ATOM 4648 N GLY B 87 −18.305 −2.578 28.911 1.00 12.00 N ATOM 4650 CA GLY B 87 −18.365 −1.984 27.589 1.00 12.48 C ATOM 4653 C GLY B 87 −17.076 −2.129 26.808 1.00 12.31 C ATOM 4654 O GLY B 87 −17.114 −2.175 25.583 1.00 14.16 O ATOM 4655 N TRP B 88 −15.931 −2.192 27.495 1.00 11.78 N ATOM 4657 CA TRP B 88 −14.658 −2.408 26.804 1.00 11.93 C ATOM 4659 CB TRP B 88 −13.432 −1.778 27.501 1.00 11.63 C ATOM 4662 CG TRP B 88 −12.253 −1.984 26.598 1.00 10.61 C ATOM 4663 CD1 TRP B 88 −11.202 −2.843 26.769 1.00 10.04 C ATOM 4665 NE1 TRP B 88 −10.404 −2.843 25.652 1.00 10.00 N ATOM 4667 CE2 TRP B 88 −10.932 −1.976 24.733 1.00 9.81 C ATOM 4668 CD2 TRP B 88 −12.106 −1.434 25.292 1.00 10.13 C ATOM 4669 CE3 TRP B 88 −12.838 −0.519 24.539 1.00 11.31 C ATOM 4671 CZ3 TRP B 88 −12.403 −0.194 23.276 1.00 12.74 C ATOM 4673 CH2 TRP B 88 −11.247 −0.752 22.749 1.00 12.39 C ATOM 4675 CZ2 TRP B 88 −10.504 −1.659 23.451 1.00 11.10 C ATOM 4677 C TRP B 88 −14.384 −3.874 26.554 1.00 13.20 C ATOM 4678 O TRP B 88 −13.795 −4.228 25.544 1.00 14.61 O ATOM 4679 N ARG B 89 −14.818 −4.742 27.456 1.00 14.97 N ATOM 4681 CA ARG B 89 −14.786 −6.157 27.135 1.00 17.45 C ATOM 4683 CB ARG B 89 −15.489 −6.978 28.216 1.00 18.59 C ATOM 4686 CG ARG B 89 −14.972 −8.407 28.352 1.00 20.17 C ATOM 4689 CD ARG B 89 −15.609 −9.163 29.496 1.00 22.60 C ATOM 4692 NE ARG B 89 −15.033 −8.796 30.790 1.00 24.39 N ATOM 4694 CZ ARG B 89 −13.948 −9.349 31.330 1.00 25.64 C ATOM 4695 NH1 ARG B 89 −13.279 −10.314 30.701 1.00 26.15 N ATOM 4698 NH2 ARG B 89 −13.524 −8.931 32.516 1.00 26.48 N ATOM 4701 C ARG B 89 −15.423 −6.339 25.731 1.00 19.17 C ATOM 4702 O ARG B 89 −15.043 −7.254 24.999 1.00 20.41 O ATOM 4703 N SER B 90 −16.345 −5.436 25.357 1.00 20.72 N ATOM 4705 CA SER B 90 −16.995 −5.405 24.034 1.00 21.67 C ATOM 4707 CB SER B 90 −18.412 −4.837 24.189 1.00 22.17 C ATOM 4710 OG SER B 90 −19.158 −5.584 25.125 1.00 23.91 O ATOM 4712 C SER B 90 −16.267 −4.630 22.917 1.00 21.74 C ATOM 4713 O SER B 90 −16.614 −4.789 21.746 1.00 23.22 O ATOM 4714 N GLY B 91 −15.307 −3.771 23.253 1.00 21.01 N ATOM 4716 CA GLY B 91 −14.547 −3.027 22.258 1.00 20.45 C ATOM 4719 C GLY B 91 −15.224 −1.724 21.881 1.00 20.20 C ATOM 4720 O GLY B 91 −14.868 −1.062 20.893 1.00 20.84 O ATOM 4721 N ASN B 92 −16.222 −1.355 22.669 1.00 19.28 N ATOM 4723 CA ASN B 92 −16.957 −0.132 22.417 1.00 18.38 C ATOM 4725 CB ASN B 92 −18.294 −0.171 23.169 1.00 17.92 C ATOM 4728 CG ASN B 92 −19.210 0.960 22.777 1.00 17.67 C ATOM 4729 OD1 ASN B 92 −18.749 2.066 22.576 1.00 15.73 O ATOM 4730 ND2 ASN B 92 −20.510 0.686 22.670 1.00 20.42 N ATOM 4733 C ASN B 92 −16.081 1.052 22.845 1.00 17.91 C ATOM 4734 O ASN B 92 −15.757 1.155 24.021 1.00 17.12 O ATOM 4735 N THR B 93 −15.667 1.909 21.902 1.00 17.67 N ATOM 4737 CA THR B 93 −14.789 3.068 22.178 1.00 17.77 C ATOM 4739 CB THR B 93 −14.457 3.902 20.885 1.00 18.92 C ATOM 4741 OG1 THR B 93 −13.708 5.097 21.206 1.00 21.52 O ATOM 4743 CG2 THR B 93 −15.718 4.434 20.237 1.00 18.95 C ATOM 4747 C THR B 93 −15.348 4.006 23.212 1.00 15.15 C ATOM 4748 O THR B 93 −14.591 4.719 23.860 1.00 14.57 O ATOM 4749 N ASN B 94 −16.676 4.042 23.349 1.00 13.37 N ATOM 4751 CA ASN B 94 −17.263 4.874 24.369 1.00 11.96 C ATOM 4753 CB ASN B 94 −18.767 5.001 24.180 1.00 12.29 C ATOM 4756 CG ASN B 94 −19.122 5.919 23.041 1.00 14.50 C ATOM 4757 OD1 ASN B 94 −18.348 6.785 22.653 1.00 16.90 O ATOM 4758 ND2 ASN B 94 −20.312 5.739 22.508 1.00 17.07 N ATOM 4761 C ASN B 94 −16.951 4.400 25.772 1.00 10.32 C ATOM 4762 O ASN B 94 −17.229 5.130 26.707 1.00 10.75 O ATOM 4763 N TYR B 95 −16.361 3.207 25.915 1.00 9.75 N ATOM 4765 CA TYR B 95 −15.992 2.654 27.219 1.00 9.39 C ATOM 4767 CB TYR B 95 −16.876 1.444 27.541 1.00 10.00 C ATOM 4770 CG TYR B 95 −18.334 1.826 27.578 1.00 10.15 C ATOM 4771 CD1 TYR B 95 −19.127 1.734 26.446 1.00 11.72 C ATOM 4773 CE1 TYR B 95 −20.466 2.105 26.467 1.00 12.50 C ATOM 4775 CZ TYR B 95 −21.008 2.602 27.625 1.00 13.24 C ATOM 4776 OH TYR B 95 −22.332 2.984 27.661 1.00 14.96 O ATOM 4778 CE2 TYR B 95 −20.243 2.720 28.762 1.00 12.98 C ATOM 4780 CD2 TYR B 95 −18.911 2.333 28.733 1.00 11.90 C ATOM 4782 C TYR B 95 −14.512 2.285 27.263 1.00 8.95 C ATOM 4783 O TYR B 95 −14.114 1.400 28.010 1.00 8.99 O ATOM 4784 N ASP B 96 −13.695 2.996 26.485 1.00 8.73 N ATOM 4786 CA ASP B 96 −12.272 2.693 26.401 1.00 8.57 C ATOM 4788 CB ASP B 96 −11.716 3.169 25.067 1.00 8.34 C ATOM 4791 CG ASP B 96 −10.298 2.720 24.829 1.00 8.28 C ATOM 4792 OD1 ASP B 96 −9.773 3.069 23.732 1.00 8.57 O ATOM 4793 OD2 ASP B 96 −9.674 2.040 25.677 1.00 8.50 O ATOM 4794 C ASP B 96 −11.510 3.314 27.580 1.00 7.91 C ATOM 4795 O ASP B 96 −11.002 4.442 27.510 1.00 8.32 O ATOM 4796 N TYR B 97 −11.479 2.567 28.671 1.00 8.03 N ATOM 4798 CA TYR B 97 −10.719 2.910 29.860 1.00 7.75 C ATOM 4800 CB TYR B 97 −11.386 3.992 30.707 1.00 7.83 C ATOM 4803 CG TYR B 97 −12.688 3.607 31.371 1.00 8.16 C ATOM 4804 CD1 TYR B 97 −13.893 3.674 30.681 1.00 8.43 C ATOM 4806 CE1 TYR B 97 −15.092 3.350 31.297 1.00 8.51 C ATOM 4808 CZ TYR B 97 −15.094 2.959 32.628 1.00 8.68 C ATOM 4809 OH TYR B 97 −16.265 2.673 33.298 1.00 9.75 O ATOM 4811 CE2 TYR B 97 −13.906 2.878 33.321 1.00 8.95 C ATOM 4813 CD2 TYR B 97 −12.719 3.205 32.697 1.00 8.55 C ATOM 4815 C TYR B 97 −10.531 1.625 30.653 1.00 7.47 C ATOM 4816 O TYR B 97 −11.168 0.607 30.383 1.00 7.98 O ATOM 4817 N GLY B 98 −9.659 1.684 31.647 1.00 7.59 N ATOM 4819 CA GLY B 98 −9.409 0.542 32.502 1.00 7.74 C ATOM 4822 C GLY B 98 −8.451 0.917 33.603 1.00 7.36 C ATOM 4823 O GLY B 98 −8.061 2.082 33.748 1.00 7.93 O ATOM 4824 N ALA B 99 −8.079 −0.080 34.390 1.00 7.67 N ATOM 4826 CA ALA B 99 −7.139 0.136 35.465 1.00 7.61 C ATOM 4828 CB ALA B 99 −7.846 0.428 36.770 1.00 8.42 C ATOM 4832 C ALA B 99 −6.207 −1.042 35.626 1.00 8.10 C ATOM 4833 O ALA B 99 −6.523 −2.172 35.222 1.00 8.34 O ATOM 4834 N ILE B 100 −5.045 −0.762 36.211 1.00 8.17 N ATOM 4836 CA ILE B 100 −4.042 −1.770 36.490 1.00 8.36 C ATOM 4838 CB ILE B 100 −2.709 −1.485 35.749 1.00 8.61 C ATOM 4840 CG1 ILE B 100 −2.941 −1.193 34.265 1.00 8.93 C ATOM 4843 CD1 ILE B 100 −1.682 −0.873 33.485 1.00 10.13 C ATOM 4847 CG2 ILE B 100 −1.738 −2.640 35.958 1.00 9.23 C ATOM 4851 C ILE B 100 −3.764 −1.741 37.982 1.00 8.16 C ATOM 4852 O ILE B 100 −3.527 −0.682 38.549 1.00 8.74 O ATOM 4853 N GLU B 101 −3.784 −2.903 38.627 1.00 8.55 N ATOM 4855 CA GLU B 101 −3.315 −3.015 40.003 1.00 8.71 C ATOM 4857 CB GLU B 101 −4.160 −4.001 40.797 1.00 9.18 C ATOM 4860 CG GLU B 101 −3.907 −3.943 42.293 1.00 10.20 C ATOM 4863 CD GLU B 101 −5.020 −4.604 43.089 1.00 10.31 C ATOM 4864 OE1 GLU B 101 −4.713 −5.401 43.998 1.00 12.33 O ATOM 4865 OE2 GLU B 101 −6.210 −4.354 42.782 1.00 11.03 O ATOM 4866 C GLU B 101 −1.858 −3.452 39.989 1.00 8.34 C ATOM 4867 O GLU B 101 −1.466 −4.253 39.161 1.00 9.30 O ATOM 4868 N LEU B 102 −1.073 −2.887 40.894 1.00 8.98 N ATOM 4870 CA LEU B 102 0.358 −3.079 40.934 1.00 8.90 C ATOM 4872 CB LEU B 102 1.068 −1.728 41.077 1.00 9.08 C ATOM 4875 CG LEU B 102 0.752 −0.722 39.978 1.00 10.14 C ATOM 4877 CD1 LEU B 102 1.517 0.561 40.225 1.00 10.89 C ATOM 4881 CD2 LEU B 102 1.034 −1.294 38.585 1.00 10.99 C ATOM 4885 C LEU B 102 0.807 −3.976 42.080 1.00 9.48 C ATOM 4886 O LEU B 102 0.168 −4.061 43.133 1.00 10.17 O ATOM 4887 N SER B 103 1.969 −4.589 41.866 1.00 9.89 N ATOM 4889 CA SER B 103 2.601 −5.483 42.828 1.00 10.79 C ATOM 4891 CB SER B 103 3.736 −6.273 42.146 1.00 11.99 C ATOM 4894 OG SER B 103 4.697 −5.398 41.584 1.00 15.05 O ATOM 4896 C SER B 103 3.183 −4.776 44.053 1.00 10.53 C ATOM 4897 O SER B 103 3.490 −5.433 45.047 1.00 11.56 O ATOM 4898 N GLU B 104 3.367 −3.464 43.962 1.00 10.04 N ATOM 4900 CA GLU B 104 3.968 −2.672 45.021 1.00 10.08 C ATOM 4902 CB GLU B 104 5.443 −2.395 44.738 1.00 10.68 C ATOM 4905 CG GLU B 104 6.259 −3.644 44.449 1.00 11.66 C ATOM 4908 CD GLU B 104 7.723 −3.350 44.233 1.00 13.72 C ATOM 4909 OE1 GLU B 104 8.329 −2.659 45.084 1.00 14.76 O ATOM 4910 OE2 GLU B 104 8.261 −3.802 43.208 1.00 19.30 O ATOM 4911 C GLU B 104 3.227 −1.351 45.093 1.00 9.84 C ATOM 4912 O GLU B 104 2.802 −0.809 44.065 1.00 10.32 O ATOM 4913 N PRO B 105 3.068 −0.805 46.291 1.00 9.86 N ATOM 4914 CA PRO B 105 2.283 0.420 46.478 1.00 10.40 C ATOM 4916 CB PRO B 105 1.878 0.324 47.944 1.00 11.33 C ATOM 4919 CG PRO B 105 3.053 −0.322 48.587 1.00 11.44 C ATOM 4922 CD PRO B 105 3.557 −1.331 47.587 1.00 10.49 C ATOM 4925 C PRO B 105 3.075 1.696 46.191 1.00 9.58 C ATOM 4926 O PRO B 105 3.227 2.576 47.035 1.00 10.04 O ATOM 4927 N ILE B 106 3.538 1.824 44.957 1.00 9.73 N ATOM 4929 CA ILE B 106 4.421 2.908 44.586 1.00 9.44 C ATOM 4931 CB ILE B 106 5.096 2.600 43.224 1.00 9.89 C ATOM 4933 CG1 ILE B 106 6.252 3.566 42.933 1.00 10.25 C ATOM 4936 CD1 ILE B 106 7.381 3.581 43.970 1.00 11.38 C ATOM 4940 CG2 ILE B 106 4.082 2.599 42.085 1.00 10.23 C ATOM 4944 C ILE B 106 3.729 4.271 44.620 1.00 9.14 C ATOM 4945 O ILE B 106 4.382 5.305 44.734 1.00 9.49 O ATOM 4946 N GLY B 107 2.407 4.287 44.541 1.00 9.13 N ATOM 4948 CA GLY B 107 1.648 5.503 44.748 1.00 9.30 C ATOM 4951 C GLY B 107 1.833 6.128 46.117 1.00 9.86 C ATOM 4952 O GLY B 107 1.627 7.326 46.279 1.00 10.75 O ATOM 4953 N ASN B 108 2.228 5.339 47.110 1.00 10.06 N ATOM 4955 CA ASN B 108 2.578 5.915 48.400 1.00 11.08 C ATOM 4957 CB ASN B 108 2.804 4.831 49.458 1.00 11.79 C ATOM 4960 CG ASN B 108 1.518 4.133 49.862 1.00 13.13 C ATOM 4961 OD1 ASN B 108 0.433 4.675 49.715 1.00 15.54 O ATOM 4962 ND2 ASN B 108 1.649 2.941 50.428 1.00 15.52 N ATOM 4965 C ASN B 108 3.799 6.809 48.340 1.00 11.38 C ATOM 4966 O ASN B 108 3.968 7.676 49.192 1.00 13.40 O ATOM 4967 N THR B 109 4.644 6.606 47.335 1.00 10.92 N ATOM 4969 CA THR B 109 5.811 7.449 47.106 1.00 11.13 C ATOM 4971 CB THR B 109 6.961 6.584 46.594 1.00 11.44 C ATOM 4973 OG1 THR B 109 7.329 5.636 47.604 1.00 13.07 O ATOM 4975 CG2 THR B 109 8.225 7.390 46.324 1.00 12.36 C ATOM 4979 C THR B 109 5.521 8.572 46.123 1.00 10.67 C ATOM 4980 O THR B 109 5.856 9.723 46.400 1.00 11.94 O ATOM 4981 N VAL B 110 4.931 8.247 44.975 1.00 9.89 N ATOM 4983 CA VAL B 110 4.771 9.245 43.921 1.00 9.99 C ATOM 4985 CB VAL B 110 4.883 8.652 42.504 1.00 9.69 C ATOM 4987 CG1 VAL B 110 6.238 8.008 42.291 1.00 10.32 C ATOM 4991 CG2 VAL B 110 3.749 7.687 42.194 1.00 9.18 C ATOM 4995 C VAL B 110 3.512 10.093 44.054 1.00 10.21 C ATOM 4996 O VAL B 110 3.434 11.153 43.425 1.00 11.23 O ATOM 4997 N GLY B 111 2.543 9.644 44.840 1.00 10.22 N ATOM 4999 CA GLY B 111 1.265 10.314 44.904 1.00 10.24 C ATOM 5002 C GLY B 111 0.334 9.866 43.803 1.00 9.73 C ATOM 5003 O GLY B 111 0.623 8.938 43.039 1.00 10.12 O ATOM 5004 N TYR B 112 −0.815 10.522 43.733 1.00 9.97 N ATOM 5006 CA TYR B 112 −1.832 10.140 42.768 1.00 9.95 C ATOM 5008 CB BTYR B 112 −2.648 8.897 43.221 0.35 10.43 C ATOM 5009 CB ATYR B 112 −2.598 8.884 43.221 0.65 10.39 C ATOM 5014 CG BTYR B 112 −2.791 8.641 44.714 0.35 11.58 C ATOM 5015 CG ATYR B 112 −3.133 8.921 44.615 0.65 11.41 C ATOM 5016 CD1 BTYR B 112 −1.797 7.973 45.428 0.35 12.47 C ATOM 5017 CD1 ATYR B 112 −2.406 8.376 45.672 0.65 13.18 C ATOM 5020 CE1 BTYR B 112 −1.935 7.713 46.789 0.35 13.56 C ATOM 5021 CE1 ATYR B 112 −2.905 8.381 46.970 0.65 15.52 C ATOM 5024 CZ BTYR B 112 −3.085 8.100 47.449 0.35 14.96 C ATOM 5025 CZ ATYR B 112 −4.144 8.931 47.209 0.65 16.29 C ATOM 5026 OH BTYR B 112 −3.215 7.838 48.796 0.35 16.64 O ATOM 5027 OH ATYR B 112 −4.641 8.940 48.492 0.65 18.51 O ATOM 5030 CE2 BTYR B 112 −4.097 8.743 46.766 0.35 14.31 C ATOM 5031 CE2 ATYR B 112 −4.894 9.467 46.174 0.65 14.98 C ATOM 5034 CD2 BTYR B 112 −3.951 9.007 45.400 0.35 12.89 C ATOM 5035 CD2 ATYR B 112 −4.382 9.459 44.880 0.65 13.02 C ATOM 5038 C TYR B 112 −2.745 11.327 42.440 1.00 9.75 C ATOM 5039 O TYR B 112 −2.730 12.363 43.110 1.00 10.71 O ATOM 5040 N PHE B 113 −3.495 11.159 41.355 1.00 9.55 N ATOM 5042 CA PHE B 113 −4.382 12.182 40.822 1.00 9.90 C ATOM 5044 CB PHE B 113 −4.592 11.952 39.321 1.00 9.79 C ATOM 5047 CG PHE B 113 −3.437 12.384 38.452 1.00 8.75 C ATOM 5048 CD1 PHE B 113 −3.520 13.562 37.714 1.00 9.30 C ATOM 5050 CE1 PHE B 113 −2.467 13.968 36.912 1.00 10.00 C ATOM 5052 CZ PHE B 113 −1.321 13.222 36.851 1.00 8.88 C ATOM 5054 CE2 PHE B 113 −1.220 12.060 37.566 1.00 8.70 C ATOM 5056 CD2 PHE B 113 −2.271 11.633 38.372 1.00 8.97 C ATOM 5058 C PHE B 113 −5.772 12.106 41.441 1.00 10.47 C ATOM 5059 O PHE B 113 −6.262 11.022 41.775 1.00 11.47 O ATOM 5060 N GLY B 114 −6.409 13.267 41.550 1.00 10.79 N ATOM 5062 CA GLY B 114 −7.854 13.333 41.663 1.00 11.39 C ATOM 5065 C GLY B 114 −8.481 13.125 40.293 1.00 10.67 C ATOM 5066 O GLY B 114 −7.801 13.207 39.265 1.00 10.78 O ATOM 5067 N TYR B 115 −9.781 12.875 40.278 1.00 10.57 N ATOM 5069 CA TYR B 115 −10.524 12.763 39.030 1.00 10.75 C ATOM 5071 CB TYR B 115 −10.346 11.382 38.379 1.00 10.71 C ATOM 5074 CG TYR B 115 −10.685 10.219 39.275 1.00 10.79 C ATOM 5075 CD1 TYR B 115 −11.988 9.716 39.338 1.00 10.63 C ATOM 5077 CE1 TYR B 115 −12.311 8.658 40.183 1.00 10.32 C ATOM 5079 CZ TYR B 115 −11.313 8.093 40.968 1.00 10.91 C ATOM 5080 OH TYR B 115 −11.581 7.056 41.831 1.00 12.42 O ATOM 5082 CE2 TYR B 115 −10.021 8.585 40.921 1.00 11.68 C ATOM 5084 CD2 TYR B 115 −9.715 9.638 40.074 1.00 11.40 C ATOM 5086 C TYR B 115 −11.983 13.069 39.319 1.00 10.14 C ATOM 5087 O TYR B 115 −12.466 12.866 40.448 1.00 11.11 O ATOM 5088 N SER B 116 −12.696 13.556 38.315 1.00 10.15 N ATOM 5090 CA SER B 116 −14.058 14.032 38.525 1.00 11.05 C ATOM 5092 CB SER B 116 −14.047 15.464 39.061 1.00 12.22 C ATOM 5095 OG SER B 116 −15.261 15.741 39.743 1.00 15.63 O ATOM 5097 C SER B 116 −14.881 13.963 37.258 1.00 10.80 C ATOM 5098 O SER B 116 −14.333 13.881 36.155 1.00 11.34 O ATOM 5099 N TYR B 117 −16.198 13.964 37.448 1.00 11.27 N ATOM 5101 CA TYR B 117 −17.167 14.054 36.366 1.00 11.28 C ATOM 5103 CB TYR B 117 −18.098 12.834 36.354 1.00 11.24 C ATOM 5106 CG TYR B 117 −19.039 12.746 37.533 1.00 11.74 C ATOM 5107 CD1 TYR B 117 −20.343 13.212 37.431 1.00 13.27 C ATOM 5109 CE1 TYR B 117 −21.221 13.145 38.509 1.00 15.32 C ATOM 5111 CZ TYR B 117 −20.788 12.637 39.708 1.00 15.51 C ATOM 5112 OH TYR B 117 −21.659 12.576 40.774 1.00 18.16 O ATOM 5114 CE2 TYR B 117 −19.494 12.174 39.841 1.00 15.41 C ATOM 5116 CD2 TYR B 117 −18.626 12.229 38.758 1.00 13.41 C ATOM 5118 C TYR B 117 −17.976 15.325 36.528 1.00 12.11 C ATOM 5119 O TYR B 117 −18.090 15.880 37.624 1.00 13.00 O ATOM 5120 N THR B 118 −18.546 15.790 35.430 1.00 12.32 N ATOM 5122 CA THR B 118 −19.471 16.915 35.476 1.00 13.14 C ATOM 5124 CB BTHR B 118 −18.839 18.242 34.989 0.35 13.58 C ATOM 5125 CB ATHR B 118 −18.853 18.174 34.815 0.65 13.85 C ATOM 5128 OG1 BTHR B 118 −17.607 18.487 35.674 0.35 14.98 O ATOM 5129 OG1 ATHR B 118 −18.864 18.025 33.391 0.65 12.42 O ATOM 5132 CG2 BTHR B 118 −19.688 19.435 35.421 0.35 13.29 C ATOM 5133 CG2 ATHR B 118 −17.368 18.339 35.127 0.65 14.99 C ATOM 5140 C THR B 118 −20.722 16.573 34.714 1.00 13.65 C ATOM 5141 O THR B 118 −20.751 15.691 33.870 1.00 14.93 O ATOM 5142 N THR B 119 −21.782 17.313 35.018 1.00 14.79 N ATOM 5144 CA THR B 119 −23.087 17.127 34.387 1.00 16.50 C ATOM 5146 CB THR B 119 −24.192 17.090 35.473 1.00 17.34 C ATOM 5148 OG1 THR B 119 −24.184 18.319 36.209 1.00 19.76 O ATOM 5150 CG2 THR B 119 −23.906 16.005 36.521 1.00 18.20 C ATOM 5154 C THR B 119 −23.412 18.233 33.389 1.00 16.89 C ATOM 5155 O THR B 119 −24.581 18.446 33.065 1.00 18.39 O ATOM 5156 N SER B 120 −22.392 18.945 32.932 1.00 15.85 N ATOM 5158 CA SER B 120 −22.568 20.023 31.976 1.00 15.52 C ATOM 5160 CB SER B 120 −22.688 21.348 32.714 1.00 17.29 C ATOM 5163 OG SER B 120 −21.566 21.555 33.538 1.00 19.16 O ATOM 5165 C SER B 120 −21.385 20.044 31.015 1.00 13.69 C ATOM 5166 O SER B 120 −20.433 19.256 31.151 1.00 13.49 O ATOM 5167 N SER B 121 −21.450 20.938 30.037 1.00 13.50 N ATOM 5169 CA SER B 121 −20.440 20.977 28.999 1.00 13.20 C ATOM 5171 CB SER B 121 −20.823 22.004 27.943 1.00 13.58 C ATOM 5174 OG SER B 121 −19.822 22.072 26.951 1.00 14.79 O ATOM 5176 C SER B 121 −19.065 21.321 29.561 1.00 12.29 C ATOM 5177 O SER B 121 −18.936 22.162 30.445 1.00 13.82 O ATOM 5178 N LEU B 122 −18.034 20.659 29.042 1.00 11.06 N ATOM 5180 CA LEU B 122 −16.653 20.994 29.362 1.00 10.63 C ATOM 5182 CB LEU B 122 −15.870 19.715 29.679 1.00 10.33 C ATOM 5185 CG LEU B 122 −16.152 19.154 31.076 1.00 11.02 C ATOM 5187 CD1 LEU B 122 −15.645 17.729 31.205 1.00 11.81 C ATOM 5191 CD2 LEU B 122 −15.557 20.038 32.139 1.00 12.56 C ATOM 5195 C LEU B 122 −15.968 21.775 28.238 1.00 10.13 C ATOM 5196 O LEU B 122 −14.775 22.042 28.324 1.00 10.57 O ATOM 5197 N VAL B 123 −16.708 22.183 27.209 1.00 10.64 N ATOM 5199 CA VAL B 123 −16.101 22.935 26.115 1.00 10.83 C ATOM 5201 CB VAL B 123 −17.123 23.312 25.017 1.00 11.49 C ATOM 5203 CG1 VAL B 123 −16.511 24.290 24.006 1.00 12.60 C ATOM 5207 CG2 VAL B 123 −17.603 22.060 24.288 1.00 12.49 C ATOM 5211 C VAL B 123 −15.439 24.192 26.669 1.00 10.32 C ATOM 5212 O VAL B 123 −16.057 24.936 27.431 1.00 11.74 O ATOM 5213 N GLY B 124 −14.189 24.416 26.283 1.00 10.03 N ATOM 5215 CA GLY B 124 −13.431 25.575 26.714 1.00 10.35 C ATOM 5218 C GLY B 124 −12.591 25.362 27.954 1.00 9.90 C ATOM 5219 O GLY B 124 −11.707 26.170 28.220 1.00 11.28 O ATOM 5220 N THR B 125 −12.851 24.311 28.726 1.00 9.67 N ATOM 5222 CA THR B 125 −12.062 24.049 29.919 1.00 9.49 C ATOM 5224 CB THR B 125 −12.709 22.900 30.695 1.00 10.53 C ATOM 5226 OG1 THR B 125 −13.998 23.310 31.178 1.00 13.20 O ATOM 5228 CG2 THR B 125 −11.907 22.498 31.922 1.00 10.73 C ATOM 5232 C THR B 125 −10.635 23.689 29.511 1.00 8.89 C ATOM 5233 O THR B 125 −10.437 22.872 28.619 1.00 9.49 O ATOM 5234 N THR B 126 −9.646 24.285 30.170 1.00 9.07 N ATOM 5236 CA THR B 126 −8.254 23.992 29.867 1.00 9.38 C ATOM 5238 CB THR B 126 −7.368 25.212 30.064 1.00 10.46 C ATOM 5240 OG1 THR B 126 −7.532 25.706 31.393 1.00 12.73 O ATOM 5242 CG2 THR B 126 −7.790 26.346 29.130 1.00 11.44 C ATOM 5246 C THR B 126 −7.731 22.819 30.679 1.00 8.61 C ATOM 5247 O THR B 126 −8.035 22.654 31.874 1.00 9.84 O ATOM 5248 N VAL B 127 −6.951 21.996 29.987 1.00 8.18 N ATOM 5250 CA VAL B 127 −6.403 20.764 30.520 1.00 8.03 C ATOM 5252 CB VAL B 127 −7.290 19.529 30.187 1.00 8.26 C ATOM 5254 CG1 VAL B 127 −8.635 19.599 30.912 1.00 9.66 C ATOM 5258 CG2 VAL B 127 −7.486 19.389 28.694 1.00 9.04 C ATOM 5262 C VAL B 127 −5.001 20.543 29.961 1.00 7.96 C ATOM 5263 O VAL B 127 −4.625 21.117 28.935 1.00 9.12 O ATOM 5264 N THR B 128 −4.259 19.675 30.630 1.00 7.81 N ATOM 5266 CA THR B 128 −2.953 19.208 30.209 1.00 7.75 C ATOM 5268 CB THR B 128 −1.953 19.374 31.362 1.00 8.07 C ATOM 5270 OG1 THR B 128 −1.843 20.762 31.705 1.00 9.24 O ATOM 5272 CG2 THR B 128 −0.549 18.864 31.006 1.00 8.88 C ATOM 5276 C THR B 128 −3.052 17.735 29.857 1.00 7.12 C ATOM 5277 O THR B 128 −3.715 16.967 30.556 1.00 7.80 O ATOM 5278 N ILE B 129 −2.385 17.340 28.775 1.00 6.77 N ATOM 5280 CA ILE B 129 −2.233 15.940 28.421 1.00 6.79 C ATOM 5282 CB ILE B 129 −2.874 15.613 27.062 1.00 7.12 C ATOM 5284 CG1 ILE B 129 −4.328 16.098 27.046 1.00 7.95 C ATOM 5287 CD1 ILE B 129 −5.076 15.825 25.764 1.00 8.74 C ATOM 5291 CG2 ILE B 129 −2.766 14.141 26.789 1.00 8.17 C ATOM 5295 C ILE B 129 −0.739 15.639 28.412 1.00 6.77 C ATOM 5296 O ILE B 129 0.001 16.217 27.603 1.00 7.27 O ATOM 5297 N SER B 130 −0.298 14.761 29.305 1.00 6.76 N ATOM 5299 CA SER B 130 1.112 14.417 29.438 1.00 6.68 C ATOM 5301 CB SER B 130 1.694 15.022 30.710 1.00 7.29 C ATOM 5304 OG SER B 130 3.097 14.906 30.734 1.00 8.01 O ATOM 5306 C SER B 130 1.250 12.911 29.453 1.00 6.73 C ATOM 5307 O SER B 130 0.517 12.224 30.158 1.00 6.79 O ATOM 5308 N GLY B 131 2.187 12.390 28.665 1.00 6.62 N ATOM 5310 CA GLY B 131 2.425 10.958 28.637 1.00 6.73 C ATOM 5313 C GLY B 131 3.640 10.604 27.804 1.00 6.56 C ATOM 5314 O GLY B 131 4.554 11.409 27.661 1.00 7.16 O ATOM 5315 N TYR B 132 3.652 9.381 27.288 1.00 6.84 N ATOM 5317 CA TYR B 132 4.858 8.737 26.740 1.00 7.12 C ATOM 5319 CB TYR B 132 5.165 7.463 27.555 1.00 7.14 C ATOM 5322 CG TYR B 132 5.728 7.832 28.917 1.00 7.14 C ATOM 5323 CD1 TYR B 132 7.087 8.103 29.060 1.00 7.37 C ATOM 5325 CE1 TYR B 132 7.614 8.520 30.265 1.00 7.89 C ATOM 5327 CZ TYR B 132 6.781 8.669 31.364 1.00 7.57 C ATOM 5328 OH TYR B 132 7.269 9.112 32.573 1.00 8.04 O ATOM 5330 CE2 TYR B 132 5.438 8.389 31.262 1.00 7.67 C ATOM 5332 CD2 TYR B 132 4.908 7.980 30.035 1.00 7.29 C ATOM 5334 C TYR B 132 4.676 8.424 25.250 1.00 6.94 C ATOM 5335 O TYR B 132 4.361 7.295 24.880 1.00 8.05 O ATOM 5336 N PRO B 133 4.874 9.411 24.378 1.00 7.25 N ATOM 5337 CA PRO B 133 4.670 9.185 22.944 1.00 7.42 C ATOM 5339 CB PRO B 133 4.628 10.594 22.368 1.00 8.13 C ATOM 5342 CG PRO B 133 5.503 11.387 23.285 1.00 8.21 C ATOM 5345 CD PRO B 133 5.210 10.826 24.655 1.00 7.47 C ATOM 5348 C PRO B 133 5.786 8.400 22.267 1.00 7.76 C ATOM 5349 O PRO B 133 6.974 8.597 22.533 1.00 8.78 O ATOM 5350 N GLY B 134 5.389 7.581 21.300 1.00 8.21 N ATOM 5352 CA GLY B 134 6.306 6.749 20.548 1.00 9.26 C ATOM 5355 C GLY B 134 7.046 7.440 19.418 1.00 9.69 C ATOM 5356 O GLY B 134 7.926 6.828 18.819 1.00 12.46 O ATOM 5357 N ASP B 135 6.718 8.697 19.134 1.00 8.82 N ATOM 5359 CA ASP B 135 7.459 9.489 18.154 1.00 9.23 C ATOM 5361 CB ASP B 135 6.533 10.305 17.243 1.00 8.88 C ATOM 5364 CG ASP B 135 5.732 11.364 17.966 1.00 8.72 C ATOM 5365 OD1 ASP B 135 5.506 11.238 19.200 1.00 8.57 O ATOM 5366 OD2 ASP B 135 5.290 12.341 17.292 1.00 9.32 O ATOM 5367 C ASP B 135 8.523 10.368 18.796 1.00 9.65 C ATOM 5368 O ASP B 135 9.102 11.216 18.121 1.00 11.42 O ATOM 5369 N LYS B 136 8.768 10.161 20.088 1.00 9.77 N ATOM 5371 CA LYS B 136 9.873 10.781 20.812 1.00 10.15 C ATOM 5373 CB LYS B 136 9.349 11.647 21.958 1.00 9.99 C ATOM 5376 CG LYS B 136 8.378 12.734 21.523 1.00 10.04 C ATOM 5379 CD LYS B 136 9.008 13.792 20.637 1.00 11.86 C ATOM 5382 CE LYS B 136 8.014 14.925 20.392 1.00 13.11 C ATOM 5385 NZ LYS B 136 8.453 15.910 19.384 1.00 15.13 N ATOM 5389 C LYS B 136 10.756 9.670 21.376 1.00 10.43 C ATOM 5390 O LYS B 136 10.432 8.491 21.280 1.00 11.37 O ATOM 5391 N THR B 137 11.881 10.042 21.976 1.00 11.03 N ATOM 5393 CA THR B 137 12.777 9.068 22.582 1.00 11.49 C ATOM 5395 CB THR B 137 13.887 9.813 23.343 1.00 12.46 C ATOM 5397 OG1 THR B 137 14.687 10.558 22.415 1.00 14.16 O ATOM 5399 CG2 THR B 137 14.865 8.837 24.040 1.00 13.85 C ATOM 5403 C THR B 137 12.010 8.169 23.536 1.00 10.34 C ATOM 5404 O THR B 137 11.257 8.654 24.378 1.00 9.84 O ATOM 5405 N ALA B 138 12.240 6.868 23.428 1.00 10.82 N ATOM 5407 CA ALA B 138 11.524 5.900 24.232 1.00 10.91 C ATOM 5409 CB ALA B 138 12.055 4.511 23.995 1.00 11.85 C ATOM 5413 C ALA B 138 11.631 6.256 25.702 1.00 10.45 C ATOM 5414 O ALA B 138 12.694 6.552 26.218 1.00 11.32 O ATOM 5415 N GLY B 139 10.503 6.194 26.378 1.00 10.16 N ATOM 5417 CA GLY B 139 10.468 6.419 27.800 1.00 9.75 C ATOM 5420 C GLY B 139 10.535 7.861 28.250 1.00 8.52 C ATOM 5421 O GLY B 139 10.669 8.089 29.441 1.00 9.08 O ATOM 5422 N THR B 140 10.421 8.829 27.340 1.00 8.37 N ATOM 5424 CA THR B 140 10.416 10.238 27.729 1.00 8.32 C ATOM 5426 CB THR B 140 11.318 11.119 26.843 1.00 9.11 C ATOM 5428 OG1 THR B 140 10.877 11.095 25.476 1.00 9.56 O ATOM 5430 CG2 THR B 140 12.768 10.611 26.900 1.00 10.40 C ATOM 5434 C THR B 140 8.987 10.774 27.783 1.00 7.64 C ATOM 5435 O THR B 140 8.120 10.378 26.991 1.00 8.11 O ATOM 5436 N GLN B 141 8.736 11.644 28.753 1.00 7.31 N ATOM 5438 CA GLN B 141 7.405 12.176 28.997 1.00 7.20 C ATOM 5440 CB GLN B 141 7.108 12.226 30.513 1.00 7.37 C ATOM 5443 CG GLN B 141 5.617 12.242 30.802 1.00 7.74 C ATOM 5446 CD GLN B 141 5.238 12.460 32.256 1.00 7.09 C ATOM 5447 OE1 GLN B 141 4.394 13.318 32.560 1.00 8.12 O ATOM 5448 NE2 GLN B 141 5.812 11.669 33.171 1.00 7.80 N ATOM 5451 C GLN B 141 7.284 13.551 28.353 1.00 7.04 C ATOM 5452 O GLN B 141 8.177 14.384 28.523 1.00 7.62 O ATOM 5453 N TRP B 142 6.180 13.771 27.652 1.00 7.07 N ATOM 5455 CA TRP B 142 5.912 14.982 26.902 1.00 7.12 C ATOM 5457 CB TRP B 142 6.063 14.717 25.387 1.00 7.52 C ATOM 5460 CG TRP B 142 7.481 14.449 24.987 1.00 7.95 C ATOM 5461 CD1 TRP B 142 8.205 13.342 25.263 1.00 7.71 C ATOM 5463 NE1 TRP B 142 9.486 13.476 24.796 1.00 8.83 N ATOM 5465 CE2 TRP B 142 9.612 14.702 24.206 1.00 8.82 C ATOM 5466 CD2 TRP B 142 8.363 15.342 24.303 1.00 8.41 C ATOM 5467 CE3 TRP B 142 8.227 16.617 23.758 1.00 9.13 C ATOM 5469 CZ3 TRP B 142 9.321 17.200 23.122 1.00 9.87 C ATOM 5471 CH2 TRP B 142 10.538 16.541 23.048 1.00 10.47 C ATOM 5473 CZ2 TRP B 142 10.703 15.288 23.568 1.00 10.08 C ATOM 5475 C TRP B 142 4.492 15.441 27.166 1.00 6.82 C ATOM 5476 O TRP B 142 3.594 14.623 27.352 1.00 7.54 O ATOM 5477 N GLN B 143 4.282 16.757 27.146 1.00 6.91 N ATOM 5479 CA GLN B 143 2.998 17.337 27.499 1.00 6.91 C ATOM 5481 CB GLN B 143 3.012 17.856 28.938 1.00 7.31 C ATOM 5484 CG GLN B 143 3.928 19.058 29.162 1.00 8.18 C ATOM 5487 CD GLN B 143 3.867 19.564 30.570 1.00 8.88 C ATOM 5488 OE1 GLN B 143 2.792 19.555 31.173 1.00 10.18 O ATOM 5489 NE2 GLN B 143 4.988 20.039 31.087 1.00 10.86 N ATOM 5492 C GLN B 143 2.599 18.460 26.561 1.00 7.08 C ATOM 5493 O GLN B 143 3.427 19.104 25.928 1.00 7.42 O ATOM 5494 N HIS B 144 1.296 18.711 26.521 1.00 7.02 N ATOM 5496 CA HIS B 144 0.706 19.829 25.804 1.00 7.09 C ATOM 5498 CB HIS B 144 0.457 19.463 24.342 1.00 7.49 C ATOM 5501 CG HIS B 144 0.061 20.617 23.491 1.00 7.31 C ATOM 5502 ND1 HIS B 144 −0.682 20.454 22.350 1.00 8.42 N ATOM 5504 CE1 HIS B 144 −0.861 21.643 21.800 1.00 8.61 C ATOM 5506 NE2 HIS B 144 −0.286 22.564 22.557 1.00 8.06 N ATOM 5508 CD2 HIS B 144 0.319 21.942 23.610 1.00 8.23 C ATOM 5510 C HIS B 144 −0.604 20.173 26.496 1.00 6.98 C ATOM 5511 O HIS B 144 −1.362 19.276 26.890 1.00 8.41 O ATOM 5512 N SER B 145 −0.878 21.463 26.640 1.00 7.81 N ATOM 5514 CA SER B 145 −2.093 21.946 27.290 1.00 7.65 C ATOM 5516 CB SER B 145 −1.755 22.780 28.522 1.00 8.72 C ATOM 5519 OG SER B 145 −1.027 22.028 29.472 1.00 9.99 O ATOM 5521 C SER B 145 −2.927 22.764 26.315 1.00 7.50 C ATOM 5522 O SER B 145 −2.406 23.304 25.338 1.00 8.51 O ATOM 5523 N GLY B 146 −4.218 22.863 26.598 1.00 7.67 N ATOM 5525 CA GLY B 146 −5.132 23.637 25.793 1.00 7.84 C ATOM 5528 C GLY B 146 −6.563 23.318 26.170 1.00 7.50 C ATOM 5529 O GLY B 146 −6.830 22.629 27.148 1.00 8.13 O ATOM 5530 N PRO B 147 −7.503 23.835 25.402 1.00 7.99 N ATOM 5531 CA PRO B 147 −8.924 23.707 25.733 1.00 8.42 C ATOM 5533 CB PRO B 147 −9.524 24.935 25.053 1.00 8.99 C ATOM 5536 CG PRO B 147 −8.686 25.096 23.804 1.00 9.17 C ATOM 5539 CD PRO B 147 −7.290 24.658 24.195 1.00 8.66 C ATOM 5542 C PRO B 147 −9.581 22.445 25.182 1.00 8.24 C ATOM 5543 O PRO B 147 −9.216 21.930 24.128 1.00 8.60 O ATOM 5544 N ILE B 148 −10.613 21.991 25.892 1.00 8.18 N ATOM 5546 CA ILE B 148 −11.532 21.003 25.349 1.00 8.21 C ATOM 5548 CB ILE B 148 −12.458 20.481 26.455 1.00 8.12 C ATOM 5550 CG1 ILE B 148 −11.654 19.795 27.570 1.00 9.56 C ATOM 5553 CD1 ILE B 148 −10.843 18.627 27.145 1.00 10.59 C ATOM 5557 CG2 ILE B 148 −13.529 19.585 25.887 1.00 8.52 C ATOM 5561 C ILE B 148 −12.338 21.662 24.222 1.00 8.21 C ATOM 5562 O ILE B 148 −12.939 22.728 24.410 1.00 9.55 O ATOM 5563 N ALA B 149 −12.348 21.019 23.064 1.00 8.67 N ATOM 5565 CA ALA B 149 −13.055 21.532 21.896 1.00 9.10 C ATOM 5567 CB ALA B 149 −12.286 21.197 20.632 1.00 9.84 C ATOM 5571 C ALA B 149 −14.476 21.013 21.771 1.00 9.41 C ATOM 5572 O ALA B 149 −15.352 21.743 21.301 1.00 10.63 O ATOM 5573 N ILE B 150 −14.684 19.747 22.136 1.00 9.20 N ATOM 5575 CA ILE B 150 −15.983 19.098 22.036 1.00 10.09 C ATOM 5577 CB ILE B 150 −16.093 18.145 20.814 1.00 10.95 C ATOM 5579 CG1 ILE B 150 −15.739 18.858 19.510 1.00 10.69 C ATOM 5582 CD1 ILE B 150 −15.768 17.974 18.271 1.00 11.51 C ATOM 5586 CG2 ILE B 150 −17.497 17.568 20.704 1.00 13.68 C ATOM 5590 C ILE B 150 −16.183 18.306 23.320 1.00 9.44 C ATOM 5591 O ILE B 150 −15.291 17.594 23.769 1.00 8.76 O ATOM 5592 N SER B 151 −17.372 18.427 23.889 1.00 10.89 N ATOM 5594 CA SER B 151 −17.765 17.741 25.101 1.00 11.77 C ATOM 5596 CB SER B 151 −18.167 18.810 26.136 1.00 12.97 C ATOM 5599 OG SER B 151 −18.512 18.242 27.381 1.00 15.09 O ATOM 5601 C SER B 151 −18.973 16.866 24.767 1.00 11.59 C ATOM 5602 O SER B 151 −20.087 17.374 24.707 1.00 14.34 O ATOM 5603 N GLU B 152 −18.761 15.581 24.505 1.00 11.06 N ATOM 5605 CA GLU B 152 −19.854 14.663 24.213 1.00 10.89 C ATOM 5607 CB BGLU B 152 −19.631 13.897 22.888 0.35 11.93 C ATOM 5608 CB AGLU B 152 −19.474 13.796 23.024 0.65 12.76 C ATOM 5613 CG BGLU B 152 −20.041 14.715 21.652 0.35 12.04 C ATOM 5614 CG AGLU B 152 −18.748 14.582 21.953 0.65 14.68 C ATOM 5619 CD BGLU B 152 −20.198 13.882 20.388 0.35 13.76 C ATOM 5620 CD AGLU B 152 −18.197 13.685 20.889 0.65 17.47 C ATOM 5621 OE1 BGLU B 152 −21.155 14.126 19.613 0.35 15.60 O ATOM 5622 OE1 AGLU B 152 −18.974 13.391 19.960 0.65 19.10 O ATOM 5623 OE2 BGLU B 152 −19.369 12.976 20.169 0.35 15.01 O ATOM 5624 OE2 AGLU B 152 −17.012 13.276 20.998 0.65 18.63 O ATOM 5625 C GLU B 152 −20.076 13.771 25.417 1.00 10.28 C ATOM 5626 O GLU B 152 −19.376 13.873 26.426 1.00 11.20 O ATOM 5627 N THR B 153 −21.057 12.893 25.338 1.00 9.81 N ATOM 5629 CA THR B 153 −21.430 12.101 26.492 1.00 10.13 C ATOM 5631 CB THR B 153 −22.622 11.232 26.129 1.00 10.71 C ATOM 5633 OG1 THR B 153 −23.706 12.086 25.751 1.00 12.66 O ATOM 5635 CG2 THR B 153 −23.106 10.417 27.332 1.00 11.52 C ATOM 5639 C THR B 153 −20.286 11.246 27.012 1.00 9.76 C ATOM 5640 O THR B 153 −20.065 11.197 28.214 1.00 10.41 O ATOM 5641 N TYR B 154 −19.588 10.574 26.108 1.00 9.41 N ATOM 5643 CA TYR B 154 −18.588 9.578 26.489 1.00 9.25 C ATOM 5645 CB TYR B 154 −18.890 8.217 25.847 1.00 9.78 C ATOM 5648 CG TYR B 154 −20.239 7.693 26.220 1.00 10.14 C ATOM 5649 CD1 TYR B 154 −20.470 7.150 27.475 1.00 10.50 C ATOM 5651 CE1 TYR B 154 −21.712 6.673 27.838 1.00 10.83 C ATOM 5653 CZ TYR B 154 −22.755 6.754 26.930 1.00 11.01 C ATOM 5654 OH TYR B 154 −24.016 6.299 27.224 1.00 12.69 O ATOM 5656 CE2 TYR B 154 −22.535 7.282 25.686 1.00 11.61 C ATOM 5658 CD2 TYR B 154 −21.293 7.762 25.344 1.00 11.24 C ATOM 5660 C TYR B 154 −17.172 9.972 26.116 1.00 8.89 C ATOM 5661 O TYR B 154 −16.233 9.282 26.532 1.00 9.04 O ATOM 5662 N LYS B 155 −17.001 11.045 25.351 1.00 9.30 N ATOM 5664 CA LYS B 155 −15.694 11.445 24.860 1.00 9.53 C ATOM 5666 CB BLYS B 155 −15.479 11.009 23.401 0.35 10.42 C ATOM 5667 CB ALYS B 155 −15.521 11.057 23.393 0.65 10.60 C ATOM 5672 CG BLYS B 155 −15.714 9.526 23.080 0.35 11.44 C ATOM 5673 CG ALYS B 155 −15.446 9.579 23.102 0.65 11.66 C ATOM 5678 CD BLYS B 155 −14.796 8.573 23.861 0.35 11.42 C ATOM 5679 CD ALYS B 155 −14.096 8.991 23.466 0.65 9.69 C ATOM 5684 CE BLYS B 155 −13.424 8.327 23.221 0.35 11.05 C ATOM 5685 CE ALYS B 155 −14.129 7.489 23.408 0.65 12.11 C ATOM 5690 NZ BLYS B 155 −12.677 7.235 23.943 0.35 10.75 N ATOM 5691 NZ ALYS B 155 −12.784 6.834 23.478 0.65 10.75 N ATOM 5698 C LYS B 155 −15.565 12.944 24.954 1.00 9.70 C ATOM 5699 O LYS B 155 −16.531 13.686 24.765 1.00 11.39 O ATOM 5700 N LEU B 156 −14.365 13.388 25.280 1.00 8.75 N ATOM 5702 CA LEU B 156 −13.957 14.757 25.042 1.00 8.77 C ATOM 5704 CB LEU B 156 −13.188 15.313 26.238 1.00 9.34 C ATOM 5707 CG LEU B 156 −13.899 15.230 27.589 1.00 9.50 C ATOM 5709 CD1 LEU B 156 −13.075 15.921 28.641 1.00 10.29 C ATOM 5713 CD2 LEU B 156 −15.313 15.818 27.545 1.00 10.26 C ATOM 5717 C LEU B 156 −13.063 14.781 23.817 1.00 8.24 C ATOM 5718 O LEU B 156 −12.322 13.817 23.555 1.00 9.48 O ATOM 5719 N GLN B 157 −13.115 15.863 23.049 1.00 7.55 N ATOM 5721 CA GLN B 157 −12.210 16.040 21.931 1.00 7.62 C ATOM 5723 CB GLN B 157 −12.926 15.991 20.589 1.00 8.06 C ATOM 5726 CG GLN B 157 −13.830 14.779 20.448 1.00 8.73 C ATOM 5729 CD GLN B 157 −14.089 14.415 19.009 1.00 9.08 C ATOM 5730 OE1 GLN B 157 −13.254 14.641 18.152 1.00 11.00 O ATOM 5731 NE2 GLN B 157 −15.236 13.811 18.749 1.00 10.76 N ATOM 5734 C GLN B 157 −11.462 17.344 22.077 1.00 7.12 C ATOM 5735 O GLN B 157 −11.942 18.287 22.701 1.00 7.80 O ATOM 5736 N TYR B 158 −10.267 17.376 21.508 1.00 7.08 N ATOM 5738 CA TYR B 158 −9.332 18.471 21.731 1.00 7.13 C ATOM 5740 CB TYR B 158 −8.677 18.365 23.128 1.00 6.96 C ATOM 5743 CG TYR B 158 −8.393 16.941 23.559 1.00 7.06 C ATOM 5744 CD1 TYR B 158 −7.326 16.222 23.034 1.00 7.19 C ATOM 5746 CE1 TYR B 158 −7.108 14.901 23.404 1.00 7.09 C ATOM 5748 CZ TYR B 158 −7.946 14.281 24.320 1.00 7.18 C ATOM 5749 OH TYR B 158 −7.750 12.979 24.733 1.00 7.90 O ATOM 5751 CE2 TYR B 158 −9.012 14.992 24.839 1.00 7.53 C ATOM 5753 CD2 TYR B 158 −9.232 16.292 24.455 1.00 7.22 C ATOM 5755 C TYR B 158 −8.280 18.442 20.641 1.00 6.81 C ATOM 5756 O TYR B 158 −8.009 17.396 20.043 1.00 7.32 O ATOM 5757 N ALA B 159 −7.670 19.601 20.393 1.00 7.32 N ATOM 5759 CA ALA B 159 −6.629 19.703 19.374 1.00 7.78 C ATOM 5761 CB ALA B 159 −6.680 21.052 18.663 1.00 8.55 C ATOM 5765 C ALA B 159 −5.229 19.451 19.903 1.00 7.90 C ATOM 5766 O ALA B 159 −4.297 19.384 19.110 1.00 8.83 O ATOM 5767 N MET B 160 −5.055 19.302 21.220 1.00 7.36 N ATOM 5769 CA MET B 160 −3.713 19.139 21.772 1.00 7.42 C ATOM 5771 CB MET B 160 −3.733 18.986 23.293 1.00 8.13 C ATOM 5774 CG MET B 160 −4.060 20.269 24.029 1.00 8.32 C ATOM 5777 SD MET B 160 −5.812 20.754 24.003 1.00 8.06 S ATOM 5778 CE MET B 160 −6.418 19.818 25.409 1.00 8.63 C ATOM 5782 C MET B 160 −3.042 17.927 21.119 1.00 7.49 C ATOM 5783 O MET B 160 −3.660 16.882 20.868 1.00 7.73 O ATOM 5784 N ASP B 161 −1.756 18.098 20.866 1.00 7.41 N ATOM 5786 CA ASP B 161 −0.986 17.115 20.130 1.00 7.72 C ATOM 5788 CB ASP B 161 0.316 17.758 19.654 1.00 8.40 C ATOM 5791 CG ASP B 161 0.065 18.961 18.781 1.00 8.51 C ATOM 5792 OD1 ASP B 161 0.577 20.072 19.078 1.00 9.92 O ATOM 5793 OD2 ASP B 161 −0.668 18.829 17.794 1.00 8.73 O ATOM 5794 C ASP B 161 −0.704 15.870 20.953 1.00 7.16 C ATOM 5795 O ASP B 161 −0.294 15.963 22.117 1.00 7.81 O ATOM 5796 N THR B 162 −0.901 14.722 20.319 1.00 7.20 N ATOM 5798 CA THR B 162 −0.669 13.420 20.924 1.00 7.17 C ATOM 5800 CB THR B 162 −1.969 12.811 21.499 1.00 7.29 C ATOM 5802 OG1 THR B 162 −2.905 12.578 20.436 1.00 7.90 O ATOM 5804 CG2 THR B 162 −2.645 13.727 22.509 1.00 8.12 C ATOM 5808 C THR B 162 −0.154 12.465 19.857 1.00 7.27 C ATOM 5809 O THR B 162 −0.332 12.693 18.664 1.00 7.57 O ATOM 5810 N TYR B 163 0.414 11.350 20.298 1.00 7.74 N ATOM 5812 CA TYR B 163 0.840 10.282 19.401 1.00 7.85 C ATOM 5814 CB TYR B 163 2.316 10.465 19.013 1.00 8.00 C ATOM 5817 CG TYR B 163 2.766 9.721 17.771 1.00 8.39 C ATOM 5818 CD1 TYR B 163 2.621 10.309 16.533 1.00 10.58 C ATOM 5820 CE1 TYR B 163 3.039 9.684 15.385 1.00 11.78 C ATOM 5822 CZ TYR B 163 3.642 8.452 15.458 1.00 11.29 C ATOM 5823 OH TYR B 163 4.037 7.861 14.280 1.00 14.10 O ATOM 5825 CE2 TYR B 163 3.807 7.835 16.689 1.00 9.46 C ATOM 5827 CD2 TYR B 163 3.390 8.484 17.833 1.00 8.33 C ATOM 5829 C TYR B 163 0.643 8.948 20.088 1.00 7.90 C ATOM 5830 O TYR B 163 0.537 8.870 21.310 1.00 8.15 O ATOM 5831 N GLY B 164 0.628 7.881 19.296 1.00 7.76 N ATOM 5833 CA GLY B 164 0.677 6.530 19.828 1.00 8.11 C ATOM 5836 C GLY B 164 1.667 6.417 20.964 1.00 7.49 C ATOM 5837 O GLY B 164 2.773 6.926 20.880 1.00 8.72 O ATOM 5838 N GLY B 165 1.262 5.708 22.009 1.00 7.45 N ATOM 5840 CA GLY B 165 1.988 5.644 23.269 1.00 7.27 C ATOM 5843 C GLY B 165 1.350 6.507 24.339 1.00 6.66 C ATOM 5844 O GLY B 165 1.461 6.214 25.531 1.00 7.23 O ATOM 5845 N GLN B 166 0.662 7.572 23.923 1.00 6.83 N ATOM 5847 CA GLN B 166 −0.003 8.463 24.859 1.00 6.61 C ATOM 5849 CB GLN B 166 0.045 9.919 24.381 1.00 6.74 C ATOM 5852 CG GLN B 166 1.451 10.489 24.459 1.00 7.33 C ATOM 5855 CD GLN B 166 1.507 11.943 24.074 1.00 6.80 C ATOM 5856 OE1 GLN B 166 1.609 12.277 22.895 1.00 7.44 O ATOM 5857 NE2 GLN B 166 1.421 12.831 25.056 1.00 8.44 N ATOM 5860 C GLN B 166 −1.429 8.054 25.211 1.00 6.58 C ATOM 5861 O GLN B 166 −2.003 8.683 26.096 1.00 6.83 O ATOM 5862 N ALA B 167 −2.023 7.043 24.587 1.00 6.87 N ATOM 5864 CA ALA B 167 −3.285 6.550 25.133 1.00 6.77 C ATOM 5866 CB ALA B 167 −3.854 5.379 24.386 1.00 7.55 C ATOM 5870 C ALA B 167 −3.038 6.162 26.587 1.00 6.83 C ATOM 5871 O ALA B 167 −1.998 5.619 26.939 1.00 7.16 O ATOM 5872 N GLY B 168 −4.029 6.461 27.410 1.00 6.56 N ATOM 5874 CA GLY B 168 −3.940 6.275 28.838 1.00 6.85 C ATOM 5877 C GLY B 168 −3.482 7.506 29.584 1.00 6.95 C ATOM 5878 O GLY B 168 −3.573 7.528 30.811 1.00 8.08 O ATOM 5879 N SER B 169 −2.983 8.524 28.883 1.00 6.66 N ATOM 5881 CA SER B 169 −2.522 9.730 29.550 1.00 6.73 C ATOM 5883 CB SER B 169 −1.937 10.743 28.561 1.00 6.97 C ATOM 5886 OG SER B 169 −0.786 10.274 27.893 1.00 7.01 O ATOM 5888 C SER B 169 −3.682 10.418 30.252 1.00 6.37 C ATOM 5889 O SER B 169 −4.809 10.457 29.735 1.00 7.00 O ATOM 5890 N PRO B 170 −3.423 11.031 31.401 1.00 6.55 N ATOM 5891 CA PRO B 170 −4.460 11.849 32.024 1.00 6.89 C ATOM 5893 CB PRO B 170 −3.857 12.207 33.376 1.00 7.40 C ATOM 5896 CG PRO B 170 −2.372 12.206 33.129 1.00 7.51 C ATOM 5899 CD PRO B 170 −2.132 11.112 32.117 1.00 7.19 C ATOM 5902 C PRO B 170 −4.681 13.102 31.183 1.00 6.85 C ATOM 5903 O PRO B 170 −3.735 13.676 30.622 1.00 7.40 O ATOM 5904 N VAL B 171 −5.937 13.524 31.132 1.00 7.09 N ATOM 5906 CA VAL B 171 −6.348 14.785 30.543 1.00 7.34 C ATOM 5908 CB VAL B 171 −7.465 14.557 29.506 1.00 7.54 C ATOM 5910 CG1 VAL B 171 −7.909 15.888 28.901 1.00 8.25 C ATOM 5914 CG2 VAL B 171 −7.031 13.593 28.430 1.00 7.81 C ATOM 5918 C VAL B 171 −6.840 15.593 31.737 1.00 7.34 C ATOM 5919 O VAL B 171 −7.955 15.357 32.214 1.00 8.22 O ATOM 5920 N PHE B 172 −5.982 16.449 32.278 1.00 7.59 N ATOM 5922 CA PHE B 172 −6.163 16.916 33.647 1.00 7.79 C ATOM 5924 CB PHE B 172 −5.221 16.170 34.623 1.00 8.27 C ATOM 5927 CG PHE B 172 −3.744 16.499 34.490 1.00 8.37 C ATOM 5928 CD1 PHE B 172 −3.131 17.378 35.375 1.00 9.16 C ATOM 5930 CE1 PHE B 172 −1.781 17.635 35.304 1.00 9.65 C ATOM 5932 CZ PHE B 172 −1.013 17.033 34.328 1.00 9.66 C ATOM 5934 CE2 PHE B 172 −1.601 16.164 33.436 1.00 9.16 C ATOM 5936 CD2 PHE B 172 −2.958 15.881 33.524 1.00 8.25 C ATOM 5938 C PHE B 172 −6.001 18.406 33.814 1.00 8.22 C ATOM 5939 O PHE B 172 −5.216 19.061 33.133 1.00 8.32 O ATOM 5940 N GLU B 173 −6.748 18.939 34.765 1.00 9.45 N ATOM 5942 CA GLU B 173 −6.530 20.289 35.261 1.00 10.25 C ATOM 5944 CB GLU B 173 −7.785 20.812 35.938 1.00 10.74 C ATOM 5947 CG GLU B 173 −8.990 20.794 35.029 1.00 11.64 C ATOM 5950 CD GLU B 173 −10.231 21.270 35.737 1.00 12.37 C ATOM 5951 OE1 GLU B 173 −10.771 22.325 35.349 1.00 13.46 O ATOM 5952 OE2 GLU B 173 −10.643 20.583 36.698 1.00 14.40 O ATOM 5953 C GLU B 173 −5.379 20.263 36.258 1.00 11.18 C ATOM 5954 O GLU B 173 −5.337 19.402 37.127 1.00 11.76 O ATOM 5955 N GLN B 174 −4.454 21.209 36.145 1.00 12.69 N ATOM 5957 CA GLN B 174 −3.289 21.244 37.026 1.00 14.03 C ATOM 5959 CB GLN B 174 −2.344 22.376 36.616 1.00 14.32 C ATOM 5962 CG GLN B 174 −1.682 22.176 35.261 1.00 14.57 C ATOM 5965 CD GLN B 174 −0.500 21.229 35.272 1.00 13.85 C ATOM 5966 OE1 GLN B 174 −0.120 20.709 34.207 1.00 14.35 O ATOM 5967 NE2 GLN B 174 0.089 20.999 36.440 1.00 13.83 N ATOM 5970 C GLN B 174 −3.670 21.420 38.499 1.00 14.98 C ATOM 5971 O GLN B 174 −3.055 20.828 39.382 1.00 15.06 O ATOM 5972 N SER B 175 −4.688 22.232 38.754 1.00 16.64 N ATOM 5974 CA SER B 175 −5.086 22.556 40.114 1.00 18.96 C ATOM 5976 CB SER B 175 −4.237 23.718 40.627 1.00 19.69 C ATOM 5979 OG SER B 175 −4.601 24.095 41.945 1.00 22.47 O ATOM 5981 C SER B 175 −6.561 22.930 40.126 1.00 19.54 C ATOM 5982 O SER B 175 −6.933 24.006 39.666 1.00 20.78 O ATOM 5983 N SER B 176 −7.400 22.039 40.644 1.00 19.58 N ATOM 5985 CA SER B 176 −8.842 22.251 40.640 1.00 20.22 C ATOM 5987 CB SER B 176 −9.468 21.458 39.495 1.00 20.96 C ATOM 5990 OG SER B 176 −10.867 21.629 39.459 1.00 23.01 O ATOM 5992 C SER B 176 −9.475 21.805 41.947 1.00 20.01 C ATOM 5993 O SER B 176 −8.995 20.878 42.599 1.00 18.97 O ATOM 5994 N SER B 177 −10.560 22.486 42.311 1.00 20.67 N ATOM 5996 CA SER B 177 −11.457 22.038 43.368 1.00 21.82 C ATOM 5998 CB SER B 177 −11.738 23.164 44.369 1.00 22.03 C ATOM 6001 OG SER B 177 −12.180 24.343 43.719 1.00 24.65 O ATOM 6003 C SER B 177 −12.749 21.547 42.706 1.00 22.02 C ATOM 6004 O SER B 177 −13.563 22.340 42.230 1.00 23.51 O ATOM 6005 N ARG B 178 −12.881 20.226 42.622 1.00 21.68 N ATOM 6007 CA ARG B 178 −14.097 19.547 42.176 1.00 20.93 C ATOM 6009 CB ARG B 178 −13.937 18.996 40.745 1.00 20.43 C ATOM 6012 CG ARG B 178 −13.783 20.018 39.627 1.00 18.45 C ATOM 6015 CD ARG B 178 −13.677 19.382 38.238 1.00 16.30 C ATOM 6018 NE ARG B 178 −13.336 20.340 37.188 1.00 15.06 N ATOM 6020 CZ ARG B 178 −14.210 20.982 36.429 1.00 15.42 C ATOM 6021 NH1 ARG B 178 −15.520 20.830 36.599 1.00 16.79 N ATOM 6024 NH2 ARG B 178 −13.766 21.800 35.487 1.00 15.73 N ATOM 6027 C ARG B 178 −14.317 18.378 43.127 1.00 21.07 C ATOM 6028 O ARG B 178 −13.498 18.130 44.007 1.00 21.95 O ATOM 6029 N THR B 179 −15.409 17.643 42.952 1.00 20.41 N ATOM 6031 CA THR B 179 −15.601 16.424 43.723 1.00 20.45 C ATOM 6033 CB THR B 179 −16.934 15.754 43.349 1.00 21.16 C ATOM 6035 OG1 THR B 179 −18.030 16.605 43.717 1.00 22.79 O ATOM 6037 CG2 THR B 179 −17.156 14.483 44.160 1.00 22.10 C ATOM 6041 C THR B 179 −14.439 15.480 43.434 1.00 19.46 C ATOM 6042 O THR B 179 −14.150 15.185 42.267 1.00 19.41 O ATOM 6043 N ASN B 180 −13.759 15.050 44.493 1.00 18.57 N ATOM 6045 CA ASN B 180 −12.593 14.162 44.410 1.00 18.41 C ATOM 6047 CB ASN B 180 −12.948 12.851 43.684 1.00 18.60 C ATOM 6050 CG ASN B 180 −11.881 11.765 43.846 1.00 18.64 C ATOM 6051 OD1 ASN B 180 −11.492 11.110 42.874 1.00 17.95 O ATOM 6052 ND2 ASN B 180 −11.407 11.572 45.071 1.00 19.48 N ATOM 6055 C ASN B 180 −11.376 14.840 43.778 1.00 17.73 C ATOM 6056 O ASN B 180 −10.477 14.160 43.272 1.00 18.18 O ATOM 6057 N CYS B 181 −11.329 16.175 43.845 1.00 18.01 N ATOM 6059 CA CYS B 181 −10.170 16.955 43.412 1.00 17.47 C ATOM 6061 CB CYS B 181 −10.365 17.519 42.007 1.00 16.55 C ATOM 6064 SG CYS B 181 −10.449 16.203 40.788 1.00 14.03 S ATOM 6065 C CYS B 181 −9.864 18.092 44.372 1.00 18.41 C ATOM 6066 O CYS B 181 −10.756 18.845 44.780 1.00 19.23 O ATOM 6067 N ASN B 182 −8.595 18.188 44.734 1.00 19.10 N ATOM 6069 CA ASN B 182 −8.057 19.316 45.475 1.00 19.72 C ATOM 6071 CB ASN B 182 −8.215 19.085 46.989 1.00 20.52 C ATOM 6074 CG ASN B 182 −7.873 20.313 47.824 0.50 21.48 C ATOM 6075 OD1 ASN B 182 −7.469 20.192 48.983 0.50 23.03 O ATOM 6076 ND2 ASN B 182 −8.051 21.498 47.248 0.50 22.48 N ATOM 6079 C ASN B 182 −6.593 19.428 45.053 1.00 19.26 C ATOM 6080 O ASN B 182 −5.683 19.248 45.854 1.00 20.75 O ATOM 6081 N GLY B 183 −6.392 19.722 43.767 1.00 17.83 N ATOM 6083 CA GLY B 183 −5.102 19.586 43.106 1.00 16.31 C ATOM 6086 C GLY B 183 −5.308 19.065 41.691 1.00 15.03 C ATOM 6087 O GLY B 183 −6.328 19.348 41.063 1.00 15.07 O ATOM 6088 N PRO B 184 −4.353 18.300 41.168 1.00 13.71 N ATOM 6089 CA PRO B 184 −4.487 17.759 39.810 1.00 12.66 C ATOM 6091 CB PRO B 184 −3.241 16.889 39.651 1.00 13.12 C ATOM 6094 CG PRO B 184 −2.259 17.444 40.640 1.00 14.51 C ATOM 6097 CD PRO B 184 −3.077 17.925 41.800 1.00 14.52 C ATOM 6100 C PRO B 184 −5.769 16.941 39.671 1.00 11.46 C ATOM 6101 O PRO B 184 −6.076 16.141 40.565 1.00 12.12 O ATOM 6102 N CYS B 185 −6.500 17.154 38.581 1.00 10.93 N ATOM 6104 CA CYS B 185 −7.851 16.641 38.453 1.00 10.37 C ATOM 6106 CB CYS B 185 −8.838 17.758 38.780 1.00 11.29 C ATOM 6109 SG CYS B 185 −10.536 17.205 38.967 1.00 13.15 S ATOM 6110 C CYS B 185 −8.095 16.139 37.046 1.00 9.18 C ATOM 6111 O CYS B 185 −8.272 16.933 36.118 1.00 9.91 O ATOM 6112 N SER B 186 −8.075 14.824 36.874 1.00 9.10 N ATOM 6114 CA SER B 186 −8.312 14.244 35.555 1.00 8.73 C ATOM 6116 CB SER B 186 −7.828 12.808 35.521 1.00 9.22 C ATOM 6119 OG SER B 186 −6.445 12.784 35.662 1.00 10.95 O ATOM 6121 C SER B 186 −9.792 14.276 35.205 1.00 8.85 C ATOM 6122 O SER B 186 −10.631 13.896 36.021 1.00 9.47 O ATOM 6123 N LEU B 187 −10.070 14.716 33.981 1.00 8.48 N ATOM 6125 CA LEU B 187 −11.417 14.803 33.438 1.00 8.48 C ATOM 6127 CB LEU B 187 −11.672 16.220 32.909 1.00 8.73 C ATOM 6130 CG LEU B 187 −11.486 17.345 33.923 1.00 9.71 C ATOM 6132 CD1 LEU B 187 −11.768 18.686 33.266 1.00 10.15 C ATOM 6136 CD2 LEU B 187 −12.372 17.141 35.147 1.00 11.50 C ATOM 6140 C LEU B 187 −11.677 13.791 32.329 1.00 8.23 C ATOM 6141 O LEU B 187 −12.828 13.573 31.958 1.00 8.64 O ATOM 6142 N ALA B 188 −10.612 13.181 31.805 1.00 8.18 N ATOM 6144 CA ALA B 188 −10.694 12.216 30.727 1.00 7.99 C ATOM 6146 CB ALA B 188 −10.950 12.910 29.397 1.00 7.96 C ATOM 6150 C ALA B 188 −9.385 11.425 30.699 1.00 7.51 C ATOM 6151 O ALA B 188 −8.414 11.769 31.366 1.00 7.88 O ATOM 6152 N VAL B 189 −9.389 10.372 29.896 1.00 7.53 N ATOM 6154 CA VAL B 189 −8.217 9.552 29.624 1.00 7.68 C ATOM 6156 CB BVAL B 189 −8.268 8.135 30.229 0.35 8.28 C ATOM 6157 CB AVAL B 189 −8.511 8.063 29.995 0.65 8.30 C ATOM 6160 CG1 BVAL B 189 −9.551 7.453 29.930 0.35 9.36 C ATOM 6161 CG1 AVAL B 189 −7.306 7.209 29.742 0.65 9.65 C ATOM 6168 CG2 BVAL B 189 −7.113 7.296 29.717 0.35 10.02 C ATOM 6169 CG2 AVAL B 189 −8.970 7.917 31.433 0.65 8.62 C ATOM 6176 C VAL B 189 −7.982 9.584 28.117 1.00 7.27 C ATOM 6177 O VAL B 189 −8.890 9.267 27.338 1.00 7.48 O ATOM 6178 N HIS B 190 −6.793 9.991 27.673 1.00 6.91 N ATOM 6180 CA HIS B 190 −6.546 10.097 26.248 1.00 6.84 C ATOM 6182 CB HIS B 190 −5.167 10.736 25.956 1.00 6.94 C ATOM 6185 CG HIS B 190 −4.917 10.787 24.504 1.00 6.68 C ATOM 6186 ND1 HIS B 190 −5.791 11.423 23.659 1.00 7.50 N ATOM 6188 CE1 HIS B 190 −5.449 11.150 22.417 1.00 7.27 C ATOM 6190 NE2 HIS B 190 −4.369 10.394 22.428 1.00 7.70 N ATOM 6192 CD2 HIS B 190 −4.006 10.160 23.732 1.00 7.53 C ATOM 6194 C HIS B 190 −6.656 8.714 25.580 1.00 6.62 C ATOM 6195 O HIS B 190 −6.168 7.735 26.122 1.00 7.01 O ATOM 6196 N THR B 191 −7.271 8.655 24.402 1.00 6.90 N ATOM 6198 CA THR B 191 −7.429 7.367 23.723 1.00 7.18 C ATOM 6200 CB THR B 191 −8.815 6.739 23.986 1.00 7.59 C ATOM 6202 OG1 THR B 191 −9.845 7.700 23.751 1.00 9.16 O ATOM 6204 CG2 THR B 191 −8.974 6.296 25.430 1.00 8.33 C ATOM 6208 C THR B 191 −7.162 7.340 22.221 1.00 7.39 C ATOM 6209 O THR B 191 −6.635 6.336 21.746 1.00 8.24 O ATOM 6210 N ASN B 192 −7.589 8.362 21.472 1.00 7.54 N ATOM 6212 CA ASN B 192 −7.637 8.270 20.016 1.00 8.61 C ATOM 6214 CB ASN B 192 −9.084 8.158 19.500 1.00 10.14 C ATOM 6217 CG ASN B 192 −9.884 7.097 20.205 1.00 13.02 C ATOM 6218 OD1 ASN B 192 −9.925 5.949 19.768 1.00 17.47 O ATOM 6219 ND2 ASN B 192 −10.571 7.484 21.269 1.00 13.77 N ATOM 6222 C ASN B 192 −7.053 9.497 19.349 1.00 7.81 C ATOM 6223 O ASN B 192 −7.187 10.604 19.845 1.00 7.54 O ATOM 6224 N GLY B 193 −6.466 9.272 18.178 1.00 7.89 N ATOM 6226 CA GLY B 193 −6.097 10.347 17.282 1.00 7.94 C ATOM 6229 C GLY B 193 −7.269 10.780 16.424 1.00 7.68 C ATOM 6230 O GLY B 193 −8.434 10.495 16.712 1.00 8.59 O ATOM 6231 N VAL B 194 −6.934 11.448 15.329 1.00 7.94 N ATOM 6233 CA VAL B 194 −7.905 12.057 14.430 1.00 8.60 C ATOM 6235 CB VAL B 194 −7.210 13.166 13.608 1.00 9.21 C ATOM 6237 CG1 VAL B 194 −8.096 13.671 12.465 1.00 10.61 C ATOM 6241 CG2 VAL B 194 −6.800 14.308 14.504 1.00 9.07 C ATOM 6245 C VAL B 194 −8.484 10.982 13.518 1.00 9.30 C ATOM 6246 O VAL B 194 −7.749 10.269 12.840 1.00 10.19 O ATOM 6247 N TYR B 195 −9.806 10.861 13.489 1.00 9.47 N ATOM 6249 CA TYR B 195 −10.480 9.922 12.601 1.00 10.28 C ATOM 6251 CB TYR B 195 −10.327 8.471 13.092 1.00 11.06 C ATOM 6254 CG TYR B 195 −11.205 8.082 14.268 1.00 11.54 C ATOM 6255 CD1 TYR B 195 −10.850 8.436 15.562 1.00 11.78 C ATOM 6257 CE1 TYR B 195 −11.625 8.075 16.647 1.00 13.23 C ATOM 6259 CZ TYR B 195 −12.799 7.391 16.439 1.00 14.48 C ATOM 6260 OH TYR B 195 −13.573 7.033 17.518 1.00 17.11 O ATOM 6262 CE2 TYR B 195 −13.188 7.044 15.160 1.00 15.20 C ATOM 6264 CD2 TYR B 195 −12.393 7.385 14.082 1.00 13.19 C ATOM 6266 C TYR B 195 −11.953 10.279 12.489 1.00 10.34 C ATOM 6267 O TYR B 195 −12.463 11.132 13.209 1.00 10.36 O ATOM 6268 N GLY B 196 −12.644 9.603 11.582 1.00 11.32 N ATOM 6270 CA GLY B 196 −14.087 9.629 11.600 1.00 11.81 C ATOM 6273 C GLY B 196 −14.742 10.932 11.216 1.00 11.29 C ATOM 6274 O GLY B 196 −15.881 11.184 11.604 1.00 12.54 O ATOM 6275 N GLY B 197 −14.038 11.749 10.452 1.00 11.14 N ATOM 6277 CA GLY B 197 −14.556 13.043 10.072 1.00 11.40 C ATOM 6280 C GLY B 197 −14.354 14.124 11.118 1.00 10.86 C ATOM 6281 O GLY B 197 −14.712 15.268 10.864 1.00 11.89 O ATOM 6282 N SER B 198 −13.756 13.794 12.260 1.00 10.19 N ATOM 6284 CA SER B 198 −13.394 14.795 13.240 1.00 9.83 C ATOM 6286 CB SER B 198 −13.303 14.175 14.624 1.00 9.77 C ATOM 6289 OG SER B 198 −12.942 15.156 15.567 1.00 9.88 O ATOM 6291 C SER B 198 −12.066 15.428 12.891 1.00 10.01 C ATOM 6292 O SER B 198 −11.212 14.812 12.266 1.00 12.17 O ATOM 6293 N SER B 199 −11.898 16.664 13.339 1.00 9.97 N ATOM 6295 CA SER B 199 −10.645 17.378 13.227 1.00 10.64 C ATOM 6297 CB SER B 199 −10.911 18.863 12.962 1.00 11.88 C ATOM 6300 OG SER B 199 −11.618 19.054 11.760 1.00 15.50 O ATOM 6302 C SER B 199 −9.791 17.257 14.486 1.00 9.48 C ATOM 6303 O SER B 199 −8.720 17.848 14.532 1.00 10.66 O ATOM 6304 N TYR B 200 −10.239 16.495 15.480 1.00 8.05 N ATOM 6306 CA TYR B 200 −9.643 16.523 16.805 1.00 7.75 C ATOM 6308 CB TYR B 200 −10.654 17.084 17.810 1.00 7.69 C ATOM 6311 CG TYR B 200 −11.101 18.490 17.511 1.00 8.31 C ATOM 6312 CD1 TYR B 200 −10.287 19.570 17.798 1.00 9.21 C ATOM 6314 CE1 TYR B 200 −10.680 20.867 17.534 1.00 10.29 C ATOM 6316 CZ TYR B 200 −11.910 21.112 16.988 1.00 10.84 C ATOM 6317 OH TYR B 200 −12.299 22.414 16.730 1.00 13.51 O ATOM 6319 CE2 TYR B 200 −12.751 20.065 16.697 1.00 11.29 C ATOM 6321 CD2 TYR B 200 −12.345 18.748 16.960 1.00 10.10 C ATOM 6323 C TYR B 200 −9.217 15.133 17.266 1.00 7.26 C ATOM 6324 O TYR B 200 −9.662 14.114 16.746 1.00 8.16 O ATOM 6325 N ASN B 201 −8.348 15.125 18.274 1.00 7.06 N ATOM 6327 CA ASN B 201 −8.042 13.952 19.084 1.00 7.16 C ATOM 6329 CB ASN B 201 −6.680 14.153 19.748 1.00 7.15 C ATOM 6332 CG ASN B 201 −5.554 14.230 18.742 1.00 7.21 C ATOM 6333 OD1 ASN B 201 −5.516 13.447 17.803 1.00 7.69 O ATOM 6334 ND2 ASN B 201 −4.644 15.175 18.926 1.00 7.72 N ATOM 6337 C ASN B 201 −9.132 13.735 20.118 1.00 7.29 C ATOM 6338 O ASN B 201 −9.912 14.647 20.394 1.00 7.53 O ATOM 6339 N ARG B 202 −9.206 12.536 20.697 1.00 6.98 N ATOM 6341 CA ARG B 202 −10.279 12.211 21.629 1.00 7.41 C ATOM 6343 CB ARG B 202 −11.383 11.355 20.995 1.00 8.88 C ATOM 6346 CG ARG B 202 −11.693 11.653 19.568 1.00 9.57 C ATOM 6349 CD ARG B 202 −12.972 11.011 19.099 1.00 10.98 C ATOM 6352 NE ARG B 202 −13.038 11.045 17.669 1.00 10.86 N ATOM 6354 CZ ARG B 202 −14.060 10.645 16.946 1.00 10.42 C ATOM 6355 NH1 ARG B 202 −15.207 10.244 17.495 1.00 11.93 N ATOM 6358 NH2 ARG B 202 −13.935 10.652 15.633 1.00 11.59 N ATOM 6361 C ARG B 202 −9.772 11.449 22.843 1.00 7.12 C ATOM 6362 O ARG B 202 −8.800 10.686 22.775 1.00 7.24 O ATOM 6363 N GLY B 203 −10.506 11.616 23.931 1.00 7.36 N ATOM 6365 CA GLY B 203 −10.273 10.888 25.156 1.00 7.64 C ATOM 6368 C GLY B 203 −11.594 10.478 25.782 1.00 7.46 C ATOM 6369 O GLY B 203 −12.600 11.167 25.693 1.00 8.95 O ATOM 6370 N THR B 204 −11.601 9.321 26.422 1.00 7.91 N ATOM 6372 CA THR B 204 −12.766 8.862 27.169 1.00 7.85 C ATOM 6374 CB THR B 204 −12.526 7.440 27.646 1.00 7.99 C ATOM 6376 OG1 THR B 204 −12.283 6.626 26.490 1.00 9.14 O ATOM 6378 CG2 THR B 204 −13.742 6.879 28.396 1.00 8.93 C ATOM 6382 C THR B 204 −13.049 9.778 28.339 1.00 7.48 C ATOM 6383 O THR B 204 −12.207 9.977 29.209 1.00 8.11 O ATOM 6384 N ARG B 205 −14.246 10.340 28.347 1.00 7.64 N ATOM 6386 CA ARG B 205 −14.673 11.241 29.393 1.00 8.00 C ATOM 6388 CB ARG B 205 −15.976 11.911 28.965 1.00 8.79 C ATOM 6391 CG ARG B 205 −16.504 12.958 29.902 1.00 8.59 C ATOM 6394 CD ARG B 205 −17.749 13.634 29.351 1.00 9.13 C ATOM 6397 NE ARG B 205 −18.197 14.685 30.247 1.00 9.66 N ATOM 6399 CZ ARG B 205 −19.108 15.593 29.932 1.00 11.19 C ATOM 6400 NH1 ARG B 205 −19.463 16.494 30.836 1.00 12.79 N ATOM 6403 NH2 ARG B 205 −19.631 15.622 28.720 1.00 12.68 N ATOM 6406 C ARG B 205 −14.893 10.499 30.697 1.00 8.17 C ATOM 6407 O ARG B 205 −15.442 9.398 30.704 1.00 8.50 O ATOM 6408 N ILE B 206 −14.511 11.107 31.803 1.00 7.90 N ATOM 6410 CA ILE B 206 −14.857 10.543 33.102 1.00 8.22 C ATOM 6412 CB ILE B 206 −13.888 10.984 34.205 1.00 8.53 C ATOM 6414 CG1 ILE B 206 −12.479 10.503 33.832 1.00 10.37 C ATOM 6417 CD1 ILE B 206 −11.395 10.782 34.838 1.00 11.44 C ATOM 6421 CG2 ILE B 206 −14.335 10.417 35.576 1.00 8.96 C ATOM 6425 C ILE B 206 −16.304 10.954 33.378 1.00 8.18 C ATOM 6426 O ILE B 206 −16.577 12.055 33.837 1.00 9.63 O ATOM 6427 N THR B 207 −17.221 10.054 33.053 1.00 8.48 N ATOM 6429 CA THR B 207 −18.633 10.182 33.409 1.00 8.64 C ATOM 6431 CB THR B 207 −19.500 9.287 32.520 1.00 9.02 C ATOM 6433 OG1 THR B 207 −19.159 7.926 32.815 1.00 9.39 O ATOM 6435 CG2 THR B 207 −19.290 9.543 31.017 1.00 9.86 C ATOM 6439 C THR B 207 −18.829 9.725 34.857 1.00 8.82 C ATOM 6440 O THR B 207 −17.906 9.220 35.505 1.00 9.18 O ATOM 6441 N LYS B 208 −20.060 9.852 35.352 1.00 9.26 N ATOM 6443 CA LYS B 208 −20.369 9.298 36.665 1.00 9.84 C ATOM 6445 CB LYS B 208 −21.833 9.519 37.046 1.00 10.99 C ATOM 6448 CG LYS B 208 −22.087 9.129 38.528 1.00 14.71 C ATOM 6451 CD LYS B 208 −23.399 9.593 39.068 1.00 16.96 C ATOM 6454 CE LYS B 208 −23.489 9.258 40.552 1.00 19.57 C ATOM 6457 NZ LYS B 208 −23.241 7.822 40.859 1.00 20.47 N ATOM 6461 C LYS B 208 −20.034 7.814 36.745 1.00 9.27 C ATOM 6462 O LYS B 208 −19.537 7.336 37.761 1.00 10.16 O ATOM 6463 N GLU B 209 −20.331 7.079 35.694 1.00 9.18 N ATOM 6465 CA GLU B 209 −20.113 5.643 35.715 1.00 9.01 C ATOM 6467 CB GLU B 209 −20.903 4.935 34.624 1.00 9.53 C ATOM 6470 CG GLU B 209 −22.414 5.046 34.816 1.00 10.25 C ATOM 6473 CD GLU B 209 −22.978 6.405 34.428 1.00 10.76 C ATOM 6474 OE1 GLU B 209 −23.862 6.914 35.155 1.00 12.33 O ATOM 6475 OE2 GLU B 209 −22.549 6.961 33.386 1.00 11.21 O ATOM 6476 C GLU B 209 −18.624 5.295 35.653 1.00 8.78 C ATOM 6477 O GLU B 209 −18.183 4.353 36.318 1.00 9.54 O ATOM 6478 N VAL B 210 −17.843 6.052 34.878 1.00 8.31 N ATOM 6480 CA VAL B 210 −16.392 5.869 34.868 1.00 8.33 C ATOM 6482 CB VAL B 210 −15.715 6.782 33.835 1.00 8.11 C ATOM 6484 CG1 VAL B 210 −14.194 6.643 33.918 1.00 8.28 C ATOM 6488 CG2 VAL B 210 −16.207 6.468 32.427 1.00 8.18 C ATOM 6492 C VAL B 210 −15.835 6.162 36.264 1.00 8.18 C ATOM 6493 O VAL B 210 −15.034 5.400 36.807 1.00 8.46 O ATOM 6494 N PHE B 211 −16.257 7.285 36.828 1.00 8.80 N ATOM 6496 CA PHE B 211 −15.865 7.717 38.169 1.00 8.95 C ATOM 6498 CB PHE B 211 −16.632 8.996 38.522 1.00 9.38 C ATOM 6501 CG PHE B 211 −16.350 9.534 39.891 1.00 10.33 C ATOM 6502 CD1 PHE B 211 −17.036 9.054 40.992 1.00 12.42 C ATOM 6504 CE1 PHE B 211 −16.794 9.562 42.250 1.00 14.20 C ATOM 6506 CZ PHE B 211 −15.867 10.570 42.422 1.00 14.22 C ATOM 6508 CE2 PHE B 211 −15.184 11.071 41.328 1.00 12.56 C ATOM 6510 CD2 PHE B 211 −15.427 10.548 40.077 1.00 10.94 C ATOM 6512 C PHE B 211 −16.144 6.610 39.183 1.00 9.01 C ATOM 6513 O PHE B 211 −15.284 6.254 39.997 1.00 9.62 O ATOM 6514 N ASP B 212 −17.341 6.057 39.145 1.00 9.24 N ATOM 6516 CA ASP B 212 −17.719 5.020 40.091 1.00 9.38 C ATOM 6518 CB ASP B 212 −19.220 4.742 40.000 1.00 10.22 C ATOM 6521 CG ASP B 212 −20.081 5.866 40.585 1.00 10.96 C ATOM 6522 OD1 ASP B 212 −19.596 6.712 41.352 1.00 12.82 O ATOM 6523 OD2 ASP B 212 −21.294 5.924 40.326 1.00 14.21 O ATOM 6524 C ASP B 212 −16.920 3.730 39.883 1.00 9.20 C ATOM 6525 O ASP B 212 −16.558 3.075 40.860 1.00 9.67 O ATOM 6526 N ASN B 213 −16.642 3.364 38.646 1.00 8.79 N ATOM 6528 CA ASN B 213 −15.823 2.182 38.386 1.00 8.75 C ATOM 6530 CB BASN B 213 −15.892 1.765 36.925 0.35 8.75 C ATOM 6531 CB AASN B 213 −15.742 1.816 36.880 0.65 8.93 C ATOM 6536 CG BASN B 213 −17.240 1.173 36.556 0.35 9.22 C ATOM 6537 CG AASN B 213 −16.833 0.837 36.379 0.65 9.63 C ATOM 6538 OD1 BASN B 213 −17.635 1.198 35.396 0.35 11.25 O ATOM 6539 OD1 AASN B 213 −17.182 0.862 35.191 0.65 11.89 O ATOM 6540 ND2 BASN B 213 −17.948 0.634 37.537 0.35 8.61 N ATOM 6541 ND2 AASN B 213 −17.315 −0.040 37.230 0.65 9.89 N ATOM 6546 C ASN B 213 −14.385 2.380 38.876 1.00 8.12 C ATOM 6547 O ASN B 213 −13.866 1.530 39.585 1.00 8.56 O ATOM 6548 N LEU B 214 −13.754 3.499 38.509 1.00 8.05 N ATOM 6550 CA LEU B 214 −12.388 3.756 38.975 1.00 8.09 C ATOM 6552 CB LEU B 214 −11.878 5.101 38.472 1.00 8.15 C ATOM 6555 CG LEU B 214 −11.645 5.232 36.974 1.00 8.61 C ATOM 6557 CD1 LEU B 214 −11.247 6.665 36.638 1.00 9.83 C ATOM 6561 CD2 LEU B 214 −10.596 4.247 36.475 1.00 9.91 C ATOM 6565 C LEU B 214 −12.321 3.712 40.498 1.00 8.01 C ATOM 6566 O LEU B 214 −11.378 3.153 41.070 1.00 8.50 O ATOM 6567 N THR B 215 −13.313 4.314 41.144 1.00 8.38 N ATOM 6569 CA THR B 215 −13.315 4.376 42.596 1.00 8.79 C ATOM 6571 CB THR B 215 −14.418 5.334 43.053 1.00 9.27 C ATOM 6573 OG1 THR B 215 −14.177 6.633 42.485 1.00 10.10 O ATOM 6575 CG2 THR B 215 −14.416 5.517 44.571 1.00 11.07 C ATOM 6579 C THR B 215 −13.481 2.979 43.209 1.00 8.80 C ATOM 6580 O THR B 215 −12.791 2.625 44.166 1.00 9.21 O ATOM 6581 N ASN B 216 −14.370 2.179 42.646 1.00 8.57 N ATOM 6583 CA ASN B 216 −14.557 0.818 43.115 1.00 8.67 C ATOM 6585 CB ASN B 216 −15.734 0.173 42.381 1.00 9.22 C ATOM 6588 CG ASN B 216 −15.982 −1.271 42.786 1.00 8.90 C ATOM 6589 OD1 ASN B 216 −15.870 −1.642 43.963 1.00 9.87 O ATOM 6590 ND2 ASN B 216 −16.303 −2.099 41.811 1.00 10.95 N ATOM 6593 C ASN B 216 −13.273 −0.002 42.920 1.00 8.34 C ATOM 6594 O ASN B 216 −12.861 −0.759 43.806 1.00 8.95 O ATOM 6595 N TRP B 217 −12.626 0.156 41.771 1.00 8.60 N ATOM 6597 CA TRP B 217 −11.442 −0.632 41.484 1.00 8.62 C ATOM 6599 CB TRP B 217 −11.051 −0.484 40.025 1.00 8.99 C ATOM 6602 CG TRP B 217 −12.086 −0.995 39.080 1.00 9.18 C ATOM 6603 CD1 TRP B 217 −13.046 −1.934 39.324 1.00 10.15 C ATOM 6605 NE1 TRP B 217 −13.804 −2.145 38.197 1.00 11.24 N ATOM 6607 CE2 TRP B 217 −13.350 −1.320 37.207 1.00 10.18 C ATOM 6608 CD2 TRP B 217 −12.272 −0.584 37.733 1.00 9.00 C ATOM 6609 CE3 TRP B 217 −11.640 0.346 36.907 1.00 9.73 C ATOM 6611 CZ3 TRP B 217 −12.074 0.488 35.602 1.00 11.01 C ATOM 6613 CH2 TRP B 217 −13.139 −0.262 35.117 1.00 11.64 C ATOM 6615 CZ2 TRP B 217 −13.799 −1.163 35.897 1.00 11.72 C ATOM 6617 C TRP B 217 −10.303 −0.253 42.431 1.00 8.36 C ATOM 6618 O TRP B 217 −9.603 −1.117 42.953 1.00 8.91 O ATOM 6619 N LYS B 218 −10.123 1.033 42.695 1.00 8.79 N ATOM 6621 CA LYS B 218 −9.100 1.444 43.625 1.00 9.20 C ATOM 6623 CB LYS B 218 −8.827 2.934 43.515 1.00 11.18 C ATOM 6626 CG LYS B 218 −9.737 3.843 44.197 1.00 15.17 C ATOM 6629 CD LYS B 218 −9.326 5.287 43.946 1.00 18.59 C ATOM 6632 CE LYS B 218 −10.240 6.273 44.642 1.00 20.56 C ATOM 6635 NZ LYS B 218 −9.920 6.379 46.090 1.00 23.12 N ATOM 6639 C LYS B 218 −9.431 0.985 45.054 1.00 9.00 C ATOM 6640 O LYS B 218 −8.543 0.568 45.790 1.00 10.38 O ATOM 6641 N ASN B 219 −10.709 1.008 45.430 1.00 8.68 N ATOM 6643 CA ASN B 219 −11.124 0.530 46.752 1.00 8.69 C ATOM 6645 CB ASN B 219 −12.545 1.004 47.075 1.00 9.62 C ATOM 6648 CG ASN B 219 −12.589 2.441 47.549 1.00 10.94 C ATOM 6649 OD1 ASN B 219 −11.678 2.906 48.223 1.00 14.01 O ATOM 6650 ND2 ASN B 219 −13.697 3.138 47.267 1.00 11.68 N ATOM 6653 C ASN B 219 −11.040 −0.980 46.901 1.00 8.48 C ATOM 6654 O ASN B 219 −11.108 −1.494 48.016 1.00 9.89 O ATOM 6655 N SER B 220 −10.884 −1.688 45.792 1.00 8.99 N ATOM 6657 CA SER B 220 −10.799 −3.141 45.786 1.00 8.99 C ATOM 6659 CB SER B 220 −11.517 −3.693 44.555 1.00 9.46 C ATOM 6662 OG SER B 220 −12.907 −3.416 44.600 1.00 9.93 O ATOM 6664 C SER B 220 −9.357 −3.642 45.795 1.00 9.37 C ATOM 6665 O SER B 220 −9.124 −4.844 45.742 1.00 10.44 O ATOM 6666 N ALA B 221 −8.377 −2.741 45.851 1.00 9.78 N ATOM 6668 CA ALA B 221 −6.981 −3.155 45.805 1.00 9.87 C ATOM 6670 CB ALA B 221 −6.068 −1.948 45.804 1.00 10.44 C ATOM 6674 C ALA B 221 −6.632 −4.065 46.968 1.00 11.09 C ATOM 6675 O ALA B 221 −7.064 −3.848 48.094 1.00 12.58 O ATOM 6676 N GLN B 222 −5.824 −5.080 46.664 1.00 11.56 N ATOM 6678 CA GLN B 222 −5.345 −6.085 47.610 1.00 13.48 C ATOM 6680 CB BGLN B 222 −5.070 −7.420 46.900 0.35 14.51 C ATOM 6681 CB AGLN B 222 −5.003 −7.403 46.863 0.65 14.16 C ATOM 6686 CG BGLN B 222 −3.617 −7.830 46.798 0.35 16.11 C ATOM 6687 CG AGLN B 222 −6.230 −8.072 46.189 0.65 12.89 C ATOM 6692 CD BGLN B 222 −3.455 −9.200 46.202 0.35 17.67 C ATOM 6693 CD AGLN B 222 −5.908 −9.289 45.310 0.65 14.84 C ATOM 6694 OE1 BGLN B 222 −4.040 −10.165 46.695 0.35 19.06 O ATOM 6695 OE1 AGLN B 222 −4.806 −9.840 45.371 0.65 18.23 O ATOM 6696 NE2 BGLN B 222 −2.655 −9.300 45.148 0.35 18.44 N ATOM 6697 NE2 AGLN B 222 −6.880 −9.712 44.495 0.65 13.26 N ATOM 6702 C GLN B 222 −4.109 −5.562 48.352 1.00 14.27 C ATOM 6703 O GLN B 222 −3.636 −6.231 49.284 1.00 17.27 O ATOM 6704 OXT GLN B 222 −3.579 −4.486 48.029 1.00 15.01 O ATOM 6705 CA CA B 301 −0.643 21.256 17.293 1.00 10.41 CA ATOM 13398 N ASP F 401 −10.088 3.418 14.402 1.00 20.15 N ATOM 13400 CA ASP F 401 −10.419 4.298 15.551 1.00 19.20 C ATOM 13402 CB ASP F 401 −11.005 3.471 16.700 1.00 20.61 C ATOM 13405 CG ASP F 401 −12.475 3.140 16.497 1.00 22.97 C ATOM 13406 OD1 ASP F 401 −13.045 2.395 17.327 1.00 26.18 O ATOM 13407 OD2 ASP F 401 −13.144 3.572 15.537 1.00 25.29 O ATOM 13408 C ASP F 401 −9.196 5.076 16.021 1.00 16.65 C ATOM 13409 O ASP F 401 −9.239 5.713 17.069 1.00 16.48 O ATOM 13412 N ALA F 402 −8.115 5.032 15.242 1.00 14.63 N ATOM 13414 CA ALA F 402 −6.897 5.780 15.549 1.00 12.75 C ATOM 13416 CB ALA F 402 −7.112 7.245 15.277 1.00 12.61 C ATOM 13420 C ALA F 402 −6.485 5.557 16.999 1.00 11.06 C ATOM 13421 O ALA F 402 −6.190 6.500 17.738 1.00 10.74 O ATOM 13422 N PHE F 403 −6.464 4.296 17.429 1.00 10.84 N ATOM 13424 CA PHE F 403 −6.076 4.009 18.798 1.00 10.34 C ATOM 13426 CB PHE F 403 −6.233 2.517 19.116 1.00 11.44 C ATOM 13429 CG PHE F 403 −7.671 2.025 19.183 1.00 12.38 C ATOM 13430 CD1 PHE F 403 −8.562 2.511 20.119 1.00 14.77 C ATOM 13432 CE1 PHE F 403 −9.880 2.048 20.187 1.00 17.09 C ATOM 13434 CZ PHE F 403 −10.309 1.064 19.322 1.00 18.48 C ATOM 13436 CE2 PHE F 403 −9.424 0.544 18.386 1.00 18.39 C ATOM 13438 CD2 PHE F 403 −8.109 1.018 18.324 1.00 16.19 C ATOM 13440 C PHE F 403 −4.626 4.428 19.018 1.00 10.00 C ATOM 13441 O PHE F 403 −3.748 4.110 18.209 1.00 12.25 O ATOM 13442 N GLU F 404 −4.372 5.130 20.116 1.00 8.64 N ATOM 13444 CA GLU F 404 −3.025 5.588 20.427 1.00 8.12 C ATOM 13446 CB GLU F 404 −2.992 7.120 20.524 1.00 7.95 C ATOM 13449 CG GLU F 404 −3.122 7.705 19.117 1.00 8.08 C ATOM 13452 CD GLU F 404 −3.043 9.212 19.009 1.00 7.71 C ATOM 13453 OE1 GLU F 404 −3.129 9.917 20.027 1.00 8.61 O ATOM 13454 OE2 GLU F 404 −2.901 9.672 17.856 1.00 8.80 O ATOM 13455 C GLU F 404 −2.442 4.854 21.637 1.00 8.22 C ATOM 13456 O GLU F 404 −2.865 3.708 21.892 1.00 8.82 O ATOM 13457 OXT GLU F 404 −1.513 5.394 22.258 1.00 8.53 O 

1-37. (canceled)
 38. A method for constructing a RP-II protease variant, wherein the variant has at least one altered property as compared to a parent RP-II protease, which method comprises: a) analyzing the three dimensional structure of the RP-II protease to identify, on the basis of an evaluation of structural considerations, at least one amino acid residue or at least one structural region of the RP-II protease, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant RP-II protease, which in comparison to the parent RP-II protease, has been modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant RP-II protease in a suitable host, and d) testing the resulting RP-II protease variant for said property.
 39. A method of producing a BLC like RP-II protease variant, wherein the variant has at least one altered property as compared to a parent BLC like RP-II protease, which method comprises: a) producing a model structure of the parent BLC like RP-II protease on the three-dimensional structure of BLC, b) comparing the model three-dimensional structure of the parent BLC like RP-II protease to the BLC structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step a) at least one structural part of the parent BLC like RP-II protease, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent BLC like RP-II protease to produce a nucleic acid sequence encoding at least one deletion or substitution of one or more amino acids at a position corresponding to said structural part, or at least one insertion of one or more amino acid residues in positions corresponding to said structural part; e) performing steps c) and d) iteratively N times, where N is an integer with the value of one or more; f) preparing the variant resulting from steps a)-e); g) testing the stability of said variant; and h) optionally repeating steps a)-g) recursively; and i) selecting a RP-II protease variant having at least one altered property as compared to the parent RP-II protease. j) expressing the modified nucleic acid sequence in a host cell to produce the variant RP-II protease; k) isolating the produced protease; l) purifying the isolated protease and m) recovering the purified RP-II protease variant.
 40. The method of claim 39, wherein step (c) identifies amino acid residue positions located at a distance of 10 Å or less to the ion-binding site of the RP-II protease parent, preferably positions located at a distance of 6 Å or less.
 41. The method of claim 39, wherein step (c) identifies amino acid residue positions in the RP-II protease parent, the modification of which provides for the removal of the ion binding site by modification of at least one of the positions identified.
 42. The method of claim 39, wherein step (c) identifies amino acid residue positions in highly mobile regions of the RP-II protease parent.
 43. A RP-II protease variant comprising at least one modification in an amino acid residue in a position located at a distance of 10 Å or less to the ion-binding site.
 44. The variant of claim 43, wherein modifications are made in at least one of the positions: 1, 2, 3, 4, 5, 6, 7, 8, 143, 144, 145, 146, 158, 159, 160, 161, 162, 194, 199, 200, and 201, where the positions refer to BLC or corresponding positions.
 45. The variant of claim 43, wherein the modification comprises the substitution of a positively charged amino acid residue with a neutral or negatively charged residue, or the substitution of a neutral residue with a negatively charged residue or the deletion of a positively charged or neutral residue.
 46. The variant of claim 43, wherein the ion binding site is removed by modification in at least one of the positions corresponding to positions 144 and or 161 of BLC.
 47. The variant of claim 43 comprising at least one modification in an amino acid residue in highly mobile regions in at least one of the positions corresponding to positions 26-31 (26, 27, 28, 29, 30, and 31); 89-91 (89, 90, and 91); 216-221 (216, 217, 218, 219, 220, and 221) of BLC.
 48. The variant of claim 43, wherein the parent is BLC and the modification comprises G30A and/or G91A.
 49. The variant of claim 43, comprising at least one modification made in mobile regions in at least one of the positions corresponding to positions 51-56, (51, 52, 53, 54, 55, 56), 88-94, (88, 89, 90, 91, 92, 93, 94), 118-122 (118, 119, 120, 121, 122), and 173-183 (173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183) of BLC.
 50. The variant of claim 43, having at least one disulfide bridge provided by modifying the amino acid residues in positions 128 and 145 in BLC or corresponding positions to Cys.
 51. The variant of claim 43, having a modified surface charge distribution in comparison to the parent RP-II protease comprising modifications in at least one of the positions corresponding to positions 7, 17, 95, 109, 143, 174, 209, 216, of BLC.
 52. The variant of claim 43, exhibiting improved stability in comparison to the parent RP-II protease comprising at substitution to Pro in at least one of the positions corresponding to positions 18, 115, 185, 269 and 293 in BLC.
 53. The variant of claim 43, comprising modifications in amino acid residues in positions corresponding to positions 1, 8, 22-35 (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35), 42-58 (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58), 82-100 (82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100), 129-135 (129, 130, 131, 132, 133, 134, 135), 141-142, 153-156 (153, 154, 155, 156), 158, 161-171 (161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171), 188-193 (188, 189, 190, 191, 192, 193), 195, 201-207 (201, 202, 203, 204, 205, 206, 207), 210, 213-214, 217 in BLC at a distance of less than 10 Å from the active site residues.
 54. The variant of claim 43, further comprising at least one of the modifications (i) amino acid residues in positions that form part of an Asn-Gly sequence being modified by deletion or substitution, preferably with Asp, Gln, Ser, Pro, Thr, or Tyr; (ii) amino acid residues in positions that occupied by a Trp being modified by substitution with Phe, Thr, Gln or Gly; (iii) amino acid residues in positions that are occupied by Glu or Asp being modified by substitution with Ala; (iv) amino acid residues in positions that are in positions that are the 1st or 2nd position following a position occupied by a Glu or Asp residue being modified by substitution with a Pro; or (v) amino acid residues in positions that are occupied by a Met being modified by deletion or substitution, preferably with Ser or Ala.
 55. An isolated polynucleotide comprising a nucleic acid sequence, which encodes for a RP-II protease variant of claim
 45. 56. The polynucleotide of claim 55, wherein the nucleic acid sequence has at least 80% homology with the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or SEQ ID NO:15.
 57. An isolated nucleic acid construct comprising the nucleic acid sequence of claim 55, operably linked to one or more control sequences capable of directing the expression of the polypeptide in a suitable expression host.
 58. A recombinant host cell comprising the nucleic acid construct of claim
 57. 59. A method for producing the RP-II variant of claim 43, comprising: a) cultivating the recombinant host cell of claim 31 under conditions conducive to the production of the RP-II protease variant; and b) recovering the variant.
 60. A detergent composition comprising the RP-II protease variant of claim
 43. 